Cell Journal (Yakhteh)

593-600

Authors: Mohammad Shafiee, Seyed Ahmad Aleyasin, Mohammad Vasei,Shahriar Semnani, Seyed Javad Mowla

Objective: MicroRNAs (miRNAs) are a class of non-coding RNAs (ncRNAs) that transcriptionally or post-transcriptionally regulate gene expression through degradation of their mRNA targets and/or translational suppression. However, there are a few reports on miRNA-mediated expression regulation of long ncRNAs (lncRNAs). We have previously reported a significant upregulation of the lncRNA SOX2OT and its intronic coding gene, SOX2, in esophageal squamous cell carcinoma (ESCC) tissue samples. In this study, we aimed to evaluate the effect of induced overexpression of miR-211 on SOX2OT and SOX2 expression in vitro. Materials and Methods: In this experimental study, we performed both bioinformatic and experimental analyses to examine whether these transcripts are regulated by miRNAs. From the list of potential candidate miRNAs, miR-211 was found to have complementary sequences to SOX2OT and SOX2 transcripts. To validate our finding experimentally, we transfected the NT-2 pluripotent cell line (an embryonal carcinoma stem cell) with an expression vector overexpressing miR-211. The expression changes of miR-211, SOX2OT, and SOX2 were then quantified by a real-time polymerase chain reaction (RT-PCR) approach. Results: Compared with mock-transfected cells, overexpression of miR-211 caused a significant down-regulation of both genes (P<0.05). Furthermore, flow-cytometry analysis revealed a significant elevation in sub-G1 cell population following ectopic expression of miR-211 in NT-2 cells. Conclusion: We report here, for the first time, the down-regulation of SOX2OT and SOX2 genes by an miRNA. Considering the vital role of SOX2OT and SOX2 genes in pluripotency and tumorigenesis, our data suggest an important and inhibitory role for miR-211 in the aforementioned processes.

608-616

Authors: Mohammad Reza Mirzaei, Mohammad Kazemi Arababadi, Malek Hossein Asadi,Seyed Javad Mowla

Objective: OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tissues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs)] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. Materials and Methods: In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR) array was employed for expression level analysis and the fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our data revealed up-regulation of HSPD1 (from HSP60 family) as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family) following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP- 90AB1 (from HSP90 family) as well as HSPA1B and HSPA6 (from HSP70 family) was down-regulated under similar conditions. Other stress-related genes showed varying expression pattern in the examined tumor cell lines. Conclusion: Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly correlated with the expression of HSP70 and HSP60 gene families. Keywords: HSPs, siRNA, Tumor Cell Lines

629-638

Authors: Nima Dehdilani,Karim Shamsasenjan,Aliakbar Movassaghpour,Parvin Akbarzadehlaleh,Bahram Amoughli Tabrizi,Hamed Parsa,Fatemeh Sabagi

Objective: Three-dimensional (3D) biomimetic nanofiber scaffolds have widespread applications in biomedical tissue engineering. They provide a suitable environment for cellular adhesion, survival, proliferation and differentiation, guide new tissue formation and development, and are one of the outstanding goals of tissue engineering. Electrospinning has recently emerged as a leading technique for producing biomimetic scaffolds with micro to nanoscale topography and a high porosity similar to the natural extracellular matrix (ECM). These scaffolds are comprised of synthetic and natural polymers for tissue engineering applications. Several kinds of cells such as human embryonic stem cells (hESCs) and mouse ESCs (mESCs) have been cultured and differentiated on nanofiber scaffolds. mESCs can be induced to differentiate into a particular cell lineage when cultured as embryoid bodies (EBs) on nano-sized scaffolds. Materials and Methods: We cultured mESCs (2500 cells/100 μl) in 96-well plates with knockout Dulbecco’s modified eagle medium (DMEM-KO) and Roswell Park Memorial Institute-1640 (RPMI-1640), both supplemented with 20% ESC grade fetal bovine serum (FBS) and essential factors in the presence of leukemia inhibitory factor (LIF). mESCs were seeded at a density of 2500 cells/100 μl onto electrospun polycaprolactone (PCL) nanofibers in 96-well plates. The control group comprised mESCs grown on tissue culture plates (TCP) at a density of 2500 cells/100 μl. Differentiation of mESCs into mouse hematopoietic stem cells (mHSCs) was performed by stem cell factor (SCF), interleukin-3 (IL-3), IL-6 and Fms-related tyrosine kinase ligand (Flt3-L) cytokines for both the PCL and TCP groups. We performed an experimental study of mESCs differentiation. Results: PCL was compared to conventional TCP for survival and differentiation of mESCs to mHSCs. There were significantly more mESCs in the PCL group. Flowcytometric analysis revealed differences in hematopoietic differentiation between the PCL and TCP culture systems. There were more CD34+ (Sca1+) and CD133+ cells subpopulations in the PCL group compared to the conventional TCP culture system. Conclusion: The nanofiber scaffold, as an effective surface, improves survival and differentiation of mESCs into mHSCs compared to gelatin coated TCP. More studies are necessary to understand how the topographical features of electrospun fibers affect cell growth and behavior. This can be achieved by designing biomimetic scaffolds for tissue engineering. Keywords: Mouse Embryonic Stem Cells, Hematopoietic Stem Cells, Nanofiber

648-658

Authors: Pouria HosseinNia, Mojtaba Tahmoorespur,Sayyed Morteza Hosseini, Mehdi Hajian,Somayeh Ostadhosseini, ohammad Reza Nasiri,Mohammad Hossein Nasr-Esfahani

Objective: This research intends to unravel the temporal expression profiles of genes involved in three developmentally important signaling pathways [transforming growth factor-β (TGF-β), fibroblast growth factor (FGF) and wingless/int (WNT)] during pre- and peri-implantation goat embryo development. Materials and Methods: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the downstream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 (D7) blastocysts and day-14 (D14) blastocysts] were separately prepared in triplicate for comparative real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the selected gene sets. Results: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-β, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-β, FGF and WNT components with renewal thereafter. Conclusion: The results suggested that TGF-β, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of pre- and periimplantation goat embryo development. Keywords: Goat, Gene Expression, TGF-β, FGF, WNT

668-677

Authors: Sam Zarbakhsh,Nasim Goudarzi,Maryam Shirmohammadi,Manouchehr Safari

Objective: Bone marrow and umbilical cord stromal cells are multipotential stem cells that have the ability to produce growth factors that play an important role in survival and generation of axons. The goal of this study was to evaluate the effects of the two different mesenchymal stem cells on peripheral nerve regeneration. Materials and Methods: In this experimental study, a 10 mm segment of the left sciatic nerve of male Wistar rats (250-300 g) was removed with a silicone tube interposed into this nerve gap. Bone marrow stromal cells (BMSCs) and human umbilical cord stromal cells (HUCSCs) were respectively obtained from rat and human. The cells were separately cultured and transplanted into the nerve gap. The sciatic nerve regeneration was evaluated by immunohistochemistry, and light and electron microscopy. Moreover, histomorphology of the gastrocnemius muscle was observed. Results: The nerve regeneration in the BMSCs and HUCSCs groups that had received the stem cells was significantly more favorable than the control group. In addition, the BMSCs group was significantly more favorable than the HUCSCs group (P<0.05). Conclusion: The results of this study suggest that both homograft BMSCs and heterograft HUCSCs may have the potential to regenerate peripheral nerve injury and transplantation of BMSCs may be more effective than HUCSCs in rat. Keywords: Bone Marrow Stromal Cells, Human Umbilical Cord Stromal Cells, Transplantation, Peripheral Nerve, Regeneration

692-700

Authors: Jin Kawata, Rui Yamaguchi, Takatoshi Yamamoto, Yasuji Ishimaru,Arisa Sakamoto,Manabu Aoki,Masafumi Kitano, Misako Umehashi, Eiji Hirose,Yasuo Yamaguchi

Objective: Neutrophils have an important role in the rapid innate immune response, and the release or active secretion of elastase from neutrophils is linked to various inflammatory responses. Purpose of this study was to determine how the human neutrophil elastase affects the interleukin-10 (IL-10) response in peripheral blood mononuclear cells (PBMC). Materials and Methods: In this prospective study, changes in IL-10 messenger RNA (mRNA) and protein expression levels in monocytes derived from human PBMCs were investigated after stimulation with human neutrophil elastase (HNE). A set of inhibitors was used for examining the pathways for IL-10 production induced by HNE. Results: Reverse transcription polymerase chain reaction (RT-PCR) showed that stimulation with HNE upregulated IL-10 mRNA expression by monocytes, while the enzyme-linked immunosorbent assay (ELISA) revealed an increase of IL-10 protein level in the culture medium. A phospholipase C inhibitor (U73122) partially blunted the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expression was significantly reduced by a protein kinase C (PKC) inhibitor (Rottlerin). A calcium chelator (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester: TMB-8) inhibited the response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment with a broad-spectrum PKC inhibitor (Ro-318425) partly blocked the response to HNE. Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNA expression. Conclusion: These results indicate that HNE mainly upregulates IL-10 mRNA expression and protein production in moncytes via a novel PKC theta/delta, although partially via the conventional PKC pathway. Keywords: Interleukin-10, PBMC, Protein kinase C

711-719

Authors: Ristic Natasa, Stevanovic Darko, Nesic Dejan,Ajdzanovic Vladimir,Rakocevic Rastko,Jaric Ivana,Milosevic Verica

Objective: The close relationship between energy metabolism, nutritional state, and reproductive physiology suggests that nutritional and metabolic disorders can disrupt normal reproductive function and fertility. Considering the importance of leptin and ghrelin effects in regulation of the hypothalamic-pituitary-gonadal axis, the objective of this study was to investigate the influence of obesity and centrally applied ghrelin on immunohistochemical appearance and quantitative morphology of the pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) producing cells in adult male rats. Materials and Methods: In this experimental study, animals were given two different diets: normal-fat (NF) and high-fat (HF), for 4 weeks, corresponding to normal and positive energy balance (n=2×14), respectively. Each group was subsequently divided into two subgroups (n=7) receiving intracerebroventricular (ICV) injections of either ghrelin [G, 1 μg/5 μL phosphate buffered saline (PBS)] or vehicle (5 μL PBS, control group) every 24 hours for five consecutive days. Results: Morphometric analyses showed that in HF control group, the percentage of FSH cells per unit volume of total pituitary gland tissue (in μm3), i.e. volume density (Vvc), was increased (P<0.05) by 9.1% in comparison with the NF controls. After ICV treatment with ghrelin, volume (Vc) and volume density (Vvc) of FSH cells in ghrelin+NF (GNF) and ghrelin+HF (GHF) groups remained unchanged in comparison with NF and HF controls. Volume of LH cells in HF control group was increased by 17% (P<0.05), but their Vvc was decreased by 8.3% (P<0.05) in comparison with NF controls. In GNF group, the volume of LH cells increased by 7% (P<0.05), in comparison with the NF controls, but in GHF group, the same parameter remained unchanged when compared with HF controls. The central application of ghrelin decreased the Vvc of LH cells only in GNF group by 38.9% (P<0.05) in comparison with the NF control animals. Conclusion: The present study has shown that obesity and repetitive ICV administration of low doses of ghrelin, in NF and HF rats, modulated the immunohistomorphometric features of gonadotrophs, indicating the importance of obesity and ghrelin in regulation of the reproductive function. Keywords: Obesity, Ghrelin, Gonadotrophs, Male, Rats

730-739

Authors: Nasrin Shojaie, Seyed Mahmood Ghaffari

Objective: Multi-drug resistance (MDR) is a controversial issue in traditional chemotherapy of aggressive cancers, including hepatocellular carcinoma. The major cause of MDR is suggested to be the aberrant activation of the main signaling pathways such as Wnt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) which have key roles in the maintenance of cancer stem cells (CSCs). Therefore, the evaluation of their alterations could be essential in chemo-resistant cancers such as Hepatocellular carcinoma. The main purpose of this study was to investigate the alteration of the mentioned pathways in the chemotherapy resistant cancer cells by assessing their major molecular parameters. Materials and Methods: In this experimental study, methylthiazol tetrazolium (MTT) assay, acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33342 staining, DNA fragmentation and colony formation methods were employed to investigate the cytotoxic effects of methotrexate (MTX) and doxorubicin (DOX) on PLC/PRF/5 cells. Moreover, the expression of 11 important genes involved in MDR was performed by semi-quantitative reverse transcriptase-polymerase chain reacti on (RT-PCR). Results: PLC/PRF/5 cells (Alexander) were sensitive to DOX and normally resistant to MTX. In addition, the results obtained from RT-PCR analysis revealed that β-catenin expression was significantly reduced and ABCG2 significantly overexpressed 4.85 and 3.34 times (P value<0.05) in DOX and MTX treated cells, respectively. Furthermore, a considerable expression of HIF-1α and p65 were detected only in MTX-resistant cells. Conclusion: Anti-cancer drugs may have more than one target in tumor cells. They not only participate in deregulation of Wnt but also alter NF-κB activation. Moreover, HIF-1α was the only anti-apoptotic protein that was significantly induced in the chemoresistant cells. Keywords: Doxorubicin, MDR, Methotrexate, NF-κB, Wnt

748-754

Authors: SezenYılmaz,Aylin Ustundag,Ozge Cemiloglu Ulker,Yalcın Duydu

Objective: Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentration range relevant to humans. The aim of this study was to investigate the protective effects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods: In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results: The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 μM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion: Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study. Keywords: Boric Acid, Boron, Comet Assay, Oxidative DNA Damage

756

Authors: Michael H. Preuß, Andreas Ziegler

Foroughmand et al. (1) have recently reported association between coronary artery disease (CAD) and two well-known single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 in subjects from South-West Iran. We doubt the validity of their findings. Genotyping was done using ARMS-PCR for rs1333049 and rs10757274 in their study. When we first looked at the genotype frequencies, we observed a substantial excess of heterozygote subjects for both SNPs. Specifically, the relative excess of heterozygosity (REH) (2), a measure for the strength of deviation from Hardy-Weinberg equilibrium (HWE), was approximately 137% for rs1333049 in controls (REH=2.3688, Table 1). In contrast, we did not observe any deviation from HWE in our own studies (3, 4). We additionally conducted a short literature search to identify other studies from Asia, which reported genotype frequencies in controls for rs1333049. These studies are summarized in table 1. None of these studies shows a deviation from HWE in their control groups (all P>0.05). In summary, only the recent study by Foroughmand and colleagues (1) shows a marked deviation from HWE in controls with this deviation observed for both reported SNPs. Possible reasons for deviations from HWE have been summarized, e.g., in Ziegler et al. (2). The most likely cause for such a strong deviation from HWE is genotyping errors, especially because genotyping by ARMS-PCR plus gel electrophoresis is prone to such errors. However, REH could also be caused by population specifics, which has been discussed by Namipashaki et al. (5). In any case, we (2) and others (5) recommend the investigation of HWE in population-based genetic association studies to improve quality and reliability of the research results.

412-421

Authors: Mahmoud Orazizadeh, Ali Khodadadi, Vahid Bayati, Sadegh Saremy,Maryam Farasat, Layasadat Khorsandi

Objective: Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, biosensors, food additives, pigments, manufacture of rubber products, and electronic materials. There are several studies about the effects of NPs on dermal fibroblast or keratinocytes, but very little attention has been directed towards adipose-derived mesenchymal stem cells (ASCs). A previous study has revealed that ZnO-NPs restricted the migration capability of ASCs. However, the potential toxicity of these NPs on ASCs is not well understood. This study intends to evaluate the effects of ZnO-NPs on subcutaneous ASCs. Materials and Methods: In this experimental study, In order to assess toxicity, we exposed rat ASCs to ZnO-NPs at concentrations of 10, 50, and 100 μg/ml for 48 hours. Toxicity was evaluated by cell morphology changes, cell viability assay, as well as apoptosis and necrosis detection. Results: ZnO-NPs concentration dependently reduced the survival rates of ASCs as revealed by the trypan blue exclusion and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium- bromide (MTT) tests. ZnO-NPs, at concentrations of 10 and 50 μg/ml, induced a significant increase in apoptotic indices as shown by the annexin V test. The concentration of 10 μg/ml of ZnO-NPs was more toxic. Conclusion: Lower concentrations of ZnO-NPs have toxic and apoptotic effects on subcutaneous ASCs. We recommend that ZnO-NPs be used with caution if there is a dermatological problem. Keywords: Nanoparticles, Mesenchymal Stromal Cells, Apoptosis

429-437

Authors: Shahnaz Razavi, Saeed Karbasi, Mohammad Morshed,Hamid Zarkesh Esfahani, Mohammad Golozar, Sedigheh Vaezifar

Objective: In this study, nano-biocomposite composed of poly (lactide-co-glycolide) (PLGA) and chitosan (CS) were electrospun through a single nozzle by dispersing the CS nano-powders in PLGA solution. The cellular behavior of human adipose derived stem cells (h-ADSCs) on random and aligned scaffolds was then evaluated. Materials and Methods: In this experimental study, the PLGA/CS scaffolds were prepared at the different ratios of 90/10, 80/20, and 70/30 (w/w) %. Morphology, cell adhesion and proliferation rate of h-ADSCs on the scaffolds were assessed using scanning electron microscope (SEM), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue staining respectively. Results: H-ADSCs seeded on the matrices indicated that the PLGA/CS composite matrix with aligned nanofibres and higher content of CS nano-powders gave significantly better performance than others in terms of cell adhesion and proliferation rate (P<0.05). Conclusion: We found that CS enhanced cell adhesion and proliferation rate, and aligned nanofibers guided cell growth along the longitudinal axis of the nanofibers, which would provide a beneficial approach for tissue engineering. Keywords: Chitosan, Mesenchymal Stem Cell, PLGA, Scaffold

451-460

Authors: Laleh Parsa Yeganeh, Reza Azarbaijani,Hossein Mousavi, Seyed Abolhassan Shahzadeh Fazeli, Mohammad Ali Amoozgar,Ghasem Hosseini Salekdeh

Objective: The bacterium Oceanimonas sp. (O. sp.) GK1 is a member of the Aeromonadaceae family and its genome represents several virulence genes involved in fish and human pathogenicity. In this original research study we aimed to identify and characterize the putative virulence factors and pathogenicity of this halotolerant marine bacterium using genome wide analysis. Materials and Methods: The genome data of O. sp. GK1 was obtained from NCBI. Comparative genomic study was done using MetaCyc database. Results: Whole genome data analysis of the O. sp. GK1 revealed that the bacterium possesses some important virulence genes (e.g. ZOT, RTX toxin, thermostable hemolysin, lateral flagella and type IV pili) which have been implicated in adhesion and biofilm formation and infection in some other pathogenic bacteria. Conclusion: This is the first report of the putative pathogenicity of O. sp.GK1. The genome wide analysis of the bacterium demonstrates the presence of virulence genes causing infectious diseases in many warm- and cold-blooded animals. Keywords: Pathogenicity, Virulence Factors, Halotolerant

471-497

Authors: Golnesa Kazemi-Oula,Soudeh Ghafouri-Fard, Maryam Beigom Mobasheri, Lobat Geranpayeh,Mohammad Hossein Modarressi

Objective: During the past decade, the importance of biomarker discovery has been highlighted in many aspects of cancer research. Biomarkers may have a role in early detection of cancer, prognosis and survival evaluation as well as drug response. Cancer-testis antigens (CTAs) have gained attention as cancer biomarkers because of their expression in a wide variety of tumors and restricted expression in testis. The aim of this study was to find putative biomarkers for breast cancer. Materials and Methods: In this applied-descriptive study, the expression of 4 CTAs, namely acrosin binding protein (ACRBP), outer dense fiber 4 (ODF4), Rhox homeobox family member 2 (RHOXF2) and spermatogenesis associated 19 (SPATA19) were analyzed at the transcript level in two breast cancer lines (MCF-7 and MDA-MB-231), 40 invasive ductal carcinoma samples and their adjacent normal tissues as well as 10 fibroadenoma samples by means of quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Results: All four genes were expressed in both cell lines. Expression of ODF4 and RHOXF2 was detected in 62.5% and 60% of breast cancer tissues but in 22.5 and 17.5% of normal tissues examined respectively. The expression of both RHOXF2 and ODF4 was upregulated in cancerous tissues compared with their normal adjacent tissues by 3.31- and 2.96-fold respectively. The expression of both genes was correlated with HER2/neu overexpression. RHOXF2 expression but not ODF4 was correlated with higher stages of tumors. However, no significant association was seen between expression patterns and estrogen and progesterone receptors status. Conclusion: ODF4 and RHOXF2 are proposed as putative breast cancer biomarkers at the transcript level. However, their expression at protein level should be evaluated in future studies. Keywords: Breast Cancer, Cancer-Testis Antigen, ODF4, RHOXF2

489-493

Authors: Mostafa Ganjuri,Jamal Moshtaghian,Kamran Ghaedi

Objective: Nanotechnology focuses on materials having at least one dimension of less than 100 nanometers. Nanomaterials such as Nanosilver (NS) have unique physical and chemical properties such as size, shape, surface charge. NS particles are thought to induce neuronal degeneration and necrosis in the brain. It has been reported that NS particles generate free radicals and oxidative stress which alters gene expression and induces apoptosis. This study was designed to evaluate whether the detrimental effect of NS particles is through the activation of Procaspase-3 during fetal neural development. Materials and Methods: In this experimental study, thirty Wistar female rats at day one of pregnancy were semi-randomly distributed into three groups of ten. Group 1, the control group, had no treatment. From day 1 to the end of pregnancy, groups 2 and 3 received 1 and 10 ppm NS respectively via drinking water. Newborn rats were sacrificed immediately after birth and their brains were dissected and kept frozen. Total RNA, extracted from brain homogenates, was reverse transcribed to cDNA. Quantitative real-time polymerase chain reaction (PCR) analysis was undertaken to estimate the expression level of Procaspase-3. Results: Developmental exposure to NS induced neurotoxicity and apoptosis. This correlated with a significant increase in Procaspase-3 expression level especially at 10 ppm NS. Conclusion: The pro-apoptotic activity of NS in cells is likely to due to the dysregulation of Procaspase-3. Keywords: Apoptosis, Brain, Blood-Brain Barrier, Procaspase-3

502-509

Authors: Iman Sadeghi,Mehrdad Behmanesh,Najmeh Ahmadian Chashmi,Mohsen Sharifi,Bahram Mohammad Soltani

Objective: Podophyllotoxin (PTOX), a natural compound in numerous plants, contains remarkable biological properties that include anti-tumor, anti-viral such as anti-human immunodeficiency virus (HIV) activities. In order to avoid its adverse effects, various compounds have been derived from PTOX. 6-methoxy PTOX (MPTOX) is one of the natural PTOX derivatives with an extra methoxy group. MPTOX is mostly isolated from the Linum species. This study has sought to determine the biological effects of MPTOX on cancer cell lines, 5637 and K562. Materials and Methods: In this experimental study, we treated the 5637 and K562 cancer cell lines with MPTOX in a dose- and time-dependent manner. Apoptosis was examined by flow cytometry and viability rate was analyzed by the MTT assay. Expressions of the tubulin (TUBB3) and topoisomerase II (TOPIIA) genes were determined by real-time polymerase chain reaction (PCR). Results: Treatment with MPTOX led to significant induction of apoptosis in cancer cells compared to control cells. Gene expression analysis showed reduced levels of TUBB3 and TOPIIA mRNA following MPTOX treatment. Conclusion: MPTOX inhibited TUBB3 and TOPIIA gene expression and subsequently induced cell death through apoptosis. These results suggested that MPTOX could be considered a potential anti-tumor agent. Keywords: Podophyllotoxin, Cancer, Apoptosis, Gene Expression, Real-Time PCR

520-531

Authors: Maryam Salavatifar,Sedigheh Zakeri,Nasim Hayati Roodbari,Navid Dinparast Djadid

Objective: The apical membrane antigen-1 (AMA-1) is considered as a promising candidate for development of a malaria vaccine against Plasmodium parasites. The correct conformation of this protein appears to be necessary for the stimulation of parasite-inhibitory responses, and these responses, in turn, seem to be antibody-mediated. Therefore, in the present investigation, we expressed the Plasmodium vivax AMA-1 (PvAMA-1) ectodomain in Escherichia coli (E. coli), purified it using standard procedures and characterized it to determine its biological activities for it to be used as a potential target for developing a protective and safe vivax malaria vaccine. Materials and Methods: In this experimental investigation, the ectodomain of PvAMA- 1 antigen (GenBank accession no. JX624741) was expressed in the E. coli M15- pQE30 expression system and purified with immobilized-metal affinity chromatography. The correct conformation of the recombinant protein was evaluated by Western blotting and indirect immunofluorescence antibody (IFA) test. In addition, the immunogenic properties of PvAMA-1 were evaluated in BALB/c mice with the purified protein emulsified in Freund’s adjuvant. Results: In the present study, the PvAMA-1 ectodomain was expressed at a high-level (65 mg/L) using a bacterial system. Reduced and non-reduced sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) as well as Western blot analysis confirmed the appropriate conformation and folding of PvAMA-1. The evaluation of immunogenic properties of PvAMA-1 showed that both T helper-1 and 2 cells (Th1 and Th2) responses were present in mice after three immunizations and persisted up to one year after the first immunization. Moreover, the antibodies raised against the recombinant PvAMA-1 in injected mice could recognize the native protein localized on P. vivax parasites. Conclusion: We demonstrate that our recombinant protein had proper conformation and folding. Also, there were common epitopes in the recombinant forms corresponding to native proteins. These results; therefore, indicate that the expressed PvAMA-1 has the potential to be used as a vivax malaria vaccine. Keywords: Malaria, Plasmodium vivax, Apical Membrane Antigen-1, Vaccine

540-546

Authors: Mahdieh Azin, Javad Mirnajafi-Zadeh,Mohammad Javan

Objective: Hippocampal insults have been observed in multiple sclerosis (MS) patients. Fibroblast growth factor-2 (FGF2) induces neurogenesis in the hippocampus and enhances the proliferation, migration and differentiation of oligodendrocyte progenitor cells (OPCs). In the current study, we have investigated the effect of FGF2 on the processes of gliotoxin induced demyelination and subsequent remyelination in the hippocampus. Materials and Methods: In this experimental study adult male Sprague-Dawley rats received either saline or lysolecithin (LPC) injections to the right hippocampi. Animals received intraperitoneal (i.p.) injections of FGF2 (5 ng/g) on days 0, 5, 12 and 26 post-LPC. Expressions of myelin basic protein (Mbp) as a marker of myelination, Olig2 as a marker of OPC proliferation, Nestin as a marker of neural progenitor cells, and glial fibrillary acidic protein (Gfap) as a marker of reactive astrocytes were investigated in the right hippocampi by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: There was reduced Mbp expression at seven days after LPC injection, increased expressions of Olig2 and Nestin, and the level of Gfap did not change. FGF2 treatment reversed the expression level of Mbp to the control, significantly enhanced the levels of Olig2 and Nestin, but did not change the level of Gfap. At day-28 post- LPC, the expression level of Mbp was higher than the control in LPC-treated animals that received FGF2. The levels of Olig2, Nestin and Gfap were at the control level in the non-treated LPC group but significantly higher in the FGF2-t reated LPC group. Conclusion: FGF2 enhanced hippocampal myelination and potentiated the recruitment of OPCs and neural stem cells (NSCs) to the lesion area. Long-term application of FGF2 might also enhance astrogliosis in the lesion site. Keywords: FGF2, Hippocampus, Neural Stem Cells

554-558

Authors: Hossein Malekzadeh,Maryam Robati,Hojatollah Yousefimanesh,Mehri Ghafourian Boroujerdnia, Reza Nadripour

Objective: Oral lichen planus (OLP) is a chronic inflammatory disease. Immunological factor may act as etiological factor. The cellular immune cells such as T cells are important in pathogenesis. Interferon gamma (IFN-γ) and interleukin 4 (IL-4) are secreted by T-helper 1 (Th1) and Th2, respectively. The aim of this study was to investigate the correlation between salivary levels of IFN-γ and IL-4 with OLP. Materials and Methods: This case control study included sixty three Iranian OLP patients who were selected from the Department of Oral Medicine of Ahvaz Jundishapur University of Medical Sciences from January to July 2013. An equal number of healthy volunteers were also selected as a control group. The OLP patients were then divided into two following sub-groups: reticular (n=30) and erythematous/ulcerative (n=33). All patients had no systemic disease and received no medication. IFN-γ and IL-4 levels in whole unstimulated saliva (WUS) were measured using the enzyme-linked immunosorbent assay (ELISA) test. Data analysis was done using t test, ANOVA, least significant difference (LSD) test, and the Kruskal-Wallis test. Results: Reticular OLP patients showed higher salivary IFN-γ (7.74 ± 0.09 pg/ml ) and IL-4 (3.876 ± 0.05 pg/ml) levels compared with the control group, indicating that difference was significant. Salivary IFN-γ/IL-4 ratio significantly increased compared with control group (P=0.042). Salivary IFN-γ and IL-4 levels between sub-groups (reticular and erythematous/ulcerative) were not significantly different (2.6 ± 0.06 and 2.3 ± 0.05, respectively, P<0.05). Conclusion: Salivary IFN-γ and IL-4 levels were increased in OLP patients. An increase of salivary IFN-γ/IL-4 ratio in OLP patients showed that Th1 might have a dominant role in the OLP pathogenesis. Keywords: Lichen Planus, Interleukin-4, Saliva, Interferon Gamma

564-568

Authors: Mohammad Fazeltabar Malekshah, Mahsa Sedighi,Kazem Parivar,Homa Mohseni Kouchesfahani,Mohamadali Bigdeli

Objective: Oximes are important materials in organic chemistry. Synparamethyl benzaldehyde oxime (toloaldoxime) is structurally similar to other oximes, hence we have studied its effects on the neonatal and adult female Balb/c mice reproductive systems in order to provide a platform for future studies on the production of female contraceptive drugs. Materials and Methods: In experimental study, we studied the effects of toloaldoxime on ovary growth and gonadal hormones of neonatal and adult Balb/c mice. A regression model for prediction was presented. Results: The effects of toloaldoxime on neonatal mice were more than adult mice. The greatest effect was on the number of Graafian follicles (59.6% in adult mice and 31.83% in neonatal mice). The least effect was on ovary weight, and blood serum levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH). Conclusion: According to the data obtained, toloaldoxime can be considered an antipregnancy substance. Keywords: Toloaldoxime, Ovary, Balb/c Mouse, Fertility

576-581

Authors: Deming Li,Ziwei Li,Xueqiang Guo, Cunshuan Xu

Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regeneration. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. Keywords: Epidermal Growth Factor, Methylation, Liver Regeneration

193-200

Authors: Najmaldin Saki,Saeid Abroun,Fatemeh Salari,Fakher Rahim,Mohammad Shahjahani,Javad Mohammadi-Asl

β-thalassemia is the most common single gene disorder worldwide, in which hemoglobin β-chain production is decreased. Today, the life expectancy of thalassemic patients is increased because of a variety of treatment methods; however treatment related complications have also increased. The most common side effect is osteoporosis, which usually occurs in early adulthood as a consequence of increased bone resorption. Increased bone resorption mainly results from factors such as delayed puberty, diabetes mellitus, hypothyroidism, ineffective hematopoiesis as well as hyperplasia of the bone marrow, parathyroid gland dysfunction, toxic effect of iron on osteoblasts, growth hormone (GH) and insulin-like growth factor-1 (IGF-1) deficiency. These factors disrupt the balance between osteoblasts and osteoclasts by interfering with various molecular mechanisms and result in decreased bone density. Given the high prevalence of osteopenia and osteoporosis in thalassemic patients and complexity of their development process, the goal of this review is to evaluate the molecular aspects involved in osteopenia and osteoporosis in thalassemic patients, which may be useful for therapeutic purposes.

211-220

Authors: Fazel Sahraneshin Samani,Marzieh Ebrahimi,Tahereh Zandieh,Reyhaneh Khoshchehreh,Mohamadreza Baghaban Eslaminejad,Nasser Aghdami,Hossein Baharvand

Objective: Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells (PMCs) as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133+ (UCB-CD133+) cells into newly-formed β-cells in vitro. Materials and Methods: This study is an experimental research. The UCB-CD133+ cells were purified by magnetic activated cell sorting (MACS) and differentiated into insulin producing cells (IPCs) in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay (ELISA) were used to determine expression and production of insulin and C-peptide at the protein level. Results: Our results demonstrated that UCB-CD133+ differentiated into IPCs. Cells in islet-like clusters with (out) co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges. Conclusion: Rat PMCs possibly affect differentiation of UCB-CD133+ cells into IPCs by increasing the number of immature β-cells.

243-252

Authors: Mohammad Mehdi Dehghan, Mohamadreza Baghaban Eslaminejad, Nader Motallebizadeh, Javad Ashrafi Halan, Leila Tagiyar, Sarang Soroori, Agbibi Nikmahzar, Mirsepehr Pedram, Abdolhossein Shahverdi,Hossein Kazemi Mehrjerdi, Sadra Izadi

Objective: Distraction osteogenesis (DO) is a surgical procedure used to generate large volumes of new bone for limb lengthening. Materials and Methods: In this animal experimental study, a 30% lengthening of the left tibia (mean distraction distance: 60.8 mm) was performed in ten adult male dogs by callus distraction after osteotomy and application of an Ilizarov fixator. Distraction was started on postoperative day seven with a distraction rate of 0.5 mm twice per day and carried out at a rate of 1.5 mm per day until the end of the study. Autologous bone marrow mesenchymal stem cells (BM-MSCs) and platelet-rich plasma (PRP) as the treatment group (n=5) or PRP alone (control group, n=5) were injected into the distracted callus at the middle and end of the distraction period. At the end of the consolidation period, the dogs were sacrificed after which computerized tomography (CT) and histomorphometric evaluations were performed. Results: Radiographic evaluationsrevealed that the amount and quality of callus formations were significantly higher in the treatment group (P<0.05). As measured by CT scan, the healing parametersin dogs of the treatment group were significantly greater (P<0.05). New bone formation in the treatment group was significantly higher (P<0.05). Conclusion: The present study showed that the transplantation of BM-MSCs positively affects early bony consolidation in DO. The use of MSCs might allow a shortened period of consolidation and therefore permit earlier device removal.

264-272

Authors: Mohammad Zandi, Musharifa Muzaffar, Syed Mohmad Shah, Manoj Kumar Singh, Prabhat Palta, Suresh Kumar Singla, Radheysham Manik, Manmohan Singh Chauhan

Objective: In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. Materials and Methods: In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. Results: The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. Conclusion: We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells.

288-295

Authors: Alireza Mohammadi, Farnoosh Attari, Vahab Babapour,Seyedeh-Nafiseh Hassani,Najmehsadat Masoudi, Abdolhossein Shahverdi,Hossein Baharvand

Objective: Embryonic germ (EG) cells are the results of reprogramming primordial germ cells (PGC) in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-β (TGFβ) and mitogen-activated protein kinase kinase (MEK) signaling pathways. Materials and Methods: In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor (SCF) as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through Gbanding technique. Results: Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 (GSK3β) and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3β molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFβ signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFβ instead of GSK3β significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities.

304-311

Authors: Shahnaz Razavi, Zahra Khosravizadeh, Hamid Bahramian,Mohammad Kazemi,

Objective: Due to the restricted potential of neural stem cells for regeneration of central nervous system (CNS) after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells (ADSCs) are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) in encapsulated human ADSCs (hADSCs) 10 and14 days after neural induction. Materials and Methods: In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol- 2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, immunocytoflourescent and realtime reverse transcriptase polymerase chain reaction (RT-PCR) analyzes 10 and 14 days after induction. Results: The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 (P<0.001) but MAP2 expression was increased. Conclusion: Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage.

322-331

Authors: Saeed Shokri,Aiob Soltani,Mahsa Kazemi,Dariush Sardari,Farshid Babapoor Mofrad

Objective: In today’s world, 2.45-GHz radio-frequency radiation (RFR) from industrial, scientific, medical, military and domestic applications is the main part of indoor-outdoor electromagnetic field exposure. Long-term effects of 2.45-GHz Wi-Fi radiation on male reproductive system was not known completely. Therefore, this study aimed to investigate the major cause of male infertility during short- and long-term exposure of Wi-Fi radiation. Materials and Methods: This is an animal experimental study, which was conducted in the Department of Anatomical Sciences, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, IRAN, from June to August 2014. Three-month-old male Wistar rats (n=27) were exposed to the 2.45 GHz radiation in a chamber with two Wi-Fi antennas on opposite walls. Animals were divided into the three following groups: I. control group (n=9) including healthy animals without any exposure to the antenna, II. 1-hour group (n=9) exposed to the 2.45 GHz Wi-Fi radiation for 1 hour per day during two months and III.7-hour group (n=9) exposed to the 2.45 GHz Wi-Fi radiation for 7 hours per day during 2 months. Sperm parameters, caspase-3 concentrations, histomorphometric changes of testis in addition to the apoptotic indexes were evaluated in the exposed and control animals. Results: Both 1-hour and 7-hour groups showed a decrease in sperm parameters in a time dependent pattern. In parallel, the number of apoptosis-positive cells and caspase-3 activity increased in the seminiferous tubules of exposed rats. The seminal vesicle weight reduced significantly in both1-hour or 7-hour groups in comparison to the control group. Conclusion: Regarding to the progressive privilege of 2.45 GHz wireless networks in our environment, we concluded that there should be a major concern regarding the timedependent exposure of whole-body to the higher frequencies of Wi-Fi networks existing in the vicinity of our living places.

339-354

Authors: Xiaguang Chen, Cunshuan Xu

Objective: To investigate the transdifferentiation relationship between eight types of liver cell during rat liver regeneration (LR). Materials and Methods: 114 healthy Sprague-Dawley (SD) rats were used in this experimental study. Eight types of liver cell were isolated and purified with percoll density gradient centrifugation and immunomagentic bead methods. Marker genes for eight types of cell were obtained by retrieving the relevant references and databases. Expression changes of markers for each cell of the eight cell types were measured using microarray. The relationships between the expression profiles of marker genes and transdifferentiation among liver cells were analyzed using bioinformatics. Liver cell transdifferentiation was predicted by comparing expression profiles of marker genes in different liver cells. Results: During LR hepatocytes (HCs) not only express hepatic oval cells (HOC) markers (including PROM1, KRT14 and LY6E), but also express biliary epithelial cell (BEC) markers (including KRT7 and KRT19); BECs express both HOC markers (including GABRP, PCNA and THY1) and HC markers such as CPS1, TAT, KRT8 and KRT18; both HC markers (KRT18, KRT8 and WT1) and BEC markers (KRT7 and KRT19) were detected in HOCs. Additionally, some HC markers were also significantly upregulated in hepatic stellate cells ( HSCs), sinusoidal endothelial cells (SECs) , Kupffer cells (KCs) and dendritic cells (DCs), mainly at 6-72 hours post partial hepatectomy (PH). Conclusion: Our findings indicate that there is a mutual transdifferentiation relationship between HC, BEC and HOC during LR, and a tendency for HSCs, SECs, KCs and DCs to transdifferentiate into HCs.

361-371

Authors: Sahar Pedram, Azadeh Mohammadirad, Mohammad Amin Rezvanfar,Mona Navaei-Nigjeh,Maryam Baeeri,Mohammad Abdollahi

Objective: Chlorpyrifos (CP) as an organophosphorus pesticide is thought to induce oxidative stress in human cells via producing reactive oxygen species (ROS) that leads to the presence of pathologic conditions due to apoptosis along with acetylcholinesterase (AChE) inhibition.This study aimed to evaluate the apoptotic effects of CP and to assess the protective potential of CeO2 nanoparticle (CNP) and sodium selenite (SSe) by measuring cascades of apoptosis, oxidative stress, inflammation, and AChE inhibition in human isolated lymphocytes. Materials and Methods: In the present experimental study, we examined the anti-oxidative and AChE activating potential of CNP and SSe in CP-treated human lymphocytes. Therefore, the lymphocytes were isolated and exposed to CP, CP+CNP, CP+SSe, and CP+CNP+SSe after a three-day incubation. Then tumor necrosis factor-alpha (TNF-α) release, myeloperoxidase (MPO) activity, thiobarbituric acid-reactive substances (TBARS) levels as inflammatory/oxidative stress indices along with AChE activity were assessed. In addition, the apoptotic process was measured by flow cytometry. Results: Results showed a significant reduction in the mortality rate, TNF-α, MPO activity, TBARS, and apoptosis rate in cells treated with CNP, SSe and their combination. Interestingly, both CNP and SSe were able to activate AChE which is inhibited by CP. The results supported the synergistic effect of CNP/SSe combination in the prevention of apoptosis along with oxidative stress and inflammatory cascade. Conclusion: CP induces apoptosis in isolated human lymphocytes via oxidative stress and inflammatory mediators. CP firstly produces ROS, which leads to membrane phospholipid damage. The beneficial effects of CNP and SSe in reduction of CP-induced apoptosis and restoring AChE inhibition relate to their anti-oxidative potentials.

379-388

Authors: Mohamed M. Abdel-Daim, Rania H. Abdou

Thallium acetate (TI) is a cumulative poison intimately accompanied by an increase in reactive oxygen species (ROS) formation that represents an important risk factor for tissue injury and malfunction. This study aims to determine the possible hepatoprotective and antioxidant effects of diallyl sulfide (DAS) from garlic and curcumin from turmeric against TI-induced liver injury and oxidative stress (OS) in rats. This in vivo animal study divided rats into six groups of 8 rats per group. The first group received saline and served as the control group. The second and third groups received DAS or curcumin only at a dose of 200 mg/kg. The fourth group received TI at a dose of 6.4 mg/kg for 5 consecutive days. The fifth and sixth groups received DAS or curcumin orally 1 hour before TI intoxication at the same dose as the second and third groups. Liver integrity serum enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT) were evaluated. Serum and liver tissue homogenate lipid peroxidation and OS biomarkers were measured. The data were analyzed by one-way ANOVA followed by Duncan’s multiple range test for post hoc analysis using SPSS version 16. TI induced marked oxidative liver damage as shown by significantly (P≤0.05) elevated serum AST, ALT, ALP, LDH and γ-GT levels. There were significant (P≤0.05) increases in serum and hepatic malondialdehyde (MDA) and serum nitric oxide (NO) as well as decreased hepatic glutathione (GSH) and catalase (CAT) activities. There were significantly (P≤0.05) less serum and hepatic superoxide dismutase (SOD) and total antioxidant capacity (TAC). Pre-treatment with DAS or curcumin ameliorated the changes in most studied biochemical parameters. DAS and curcumin effectively reduced TI-induced liver toxicity.

7-14

Authors: Khadijeh Falahzadeh , Amir Banaei-Esfahani , Maryam Shahhoseini

Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus.

27-36

Authors: Kazem Parivar , Javad Baharara , Azar Sheikholeslami

Objective: There are several factors, like environmental agents, neurotrophic factors, serotonin and some hormones such as estrogen, affecting neurogenesis and neural differentiation. Regarding to importance of proliferation and regeneration in central nervous system, and a progressive increase in neurodegenerative diseases, cell therapy is an attractive approach in neuroscience. The aim of the present study was to investigate the effects of sex steroid hormones and basic fibroblast growth factor (bFGF) on neuronal differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). Materials and Methods: This experimental study was established in Kharazmi University. BM was isolated from the bones of femur and tibia of 4-6-week old Naval Medical Research Institute (NMRI) mice, and the cells were cultured. The cells were divided into following 4 groups based on the applied treatments: I. control (no treatment), II. steroid hormones (β-estradiol, progesterone and testosterone), III. bFGF and IV. combination of steroid hormones and bFGF. Immunocytochemistry and flow cytometery analyses were applied for beta III-tubulin (β-III tubulin) and microtubule-associated proteins-2 (MAP-2) in 4 days of treatment for all groups. Results: The cells treated with combination of bFGF and steroid hormones represented more expressions of neural markers as compared to control and to other two groups treated with either bFGF or steroid hormones. Conclusion: This study showed that BM-MSCs can express specific neural markers after receiving bFGF pretreatment that was followed by sex steroid hormones treatment. More investigations are necessary to specify whether steroid hormones and bFGF can be considered for treatment of CNS diseases and disorders.

49-58

Authors: Saeed Shafieyan , Ehsan Taghiabadi , Parvaneh Mohammadi , Nasser Aghdami , Nasrin Falah , Zahra Orouji , Abdoreza Nazari

Objective: Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can’t be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue (FG) was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. Materials and Methods: In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin (H&E) and Masson’s Trichrome (MT) staining of the biopsies after 8 weeks. Results: Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. Conclusion: These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate.

71-82

Authors: Majid Farshdousti Hagh , Mehrdad Noruzinia , Yousef Mortazavi , Masood Soleimani , Saeed Kaviani , Saeed Abroun , Ali Dehghani Fard , Maryam Mahmoodinia Maymand

Objective: Runt-related transcription factor 2 (RUNX2) and osterix (OSX) as two specific osteoblast transcription factors and distal-less homeobox 5 (DLX5) as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein (BSP), osteopontin and collagen type Iα1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells (MSCs). Materials and Methods: In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR (MSP). Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction (PCR). Results: MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. Conclusion: The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role.

89-98

Authors: Ali Mohammad Foroughmand , Emad Nikkhah , Hamid Galehdari , Mohammad Hossin Jadbabaee

Objective: Coronary artery disease (CAD) is a multi-factorial and heterogenic disease with atherosclerosis plaques formation in internal wall of coronary artery. Plaque formation results to limitation of the blood reaching to myocardium leading to appearance of some problems, such as ischemia, sudden thrombosis veins and myocardial infarction (MI). Several environmental and genetic factors are involved in prevalence and incident of CAD as follows: hypertension, high low density lipoprotein-cholesterol (LDL-C), age, diabetes mellitus, family history of early-onset heart disease and smoking. According to genome wide association studies (GWAS), five polymorphisms in the 9p21 locus seem to be associated with the CAD. We aimed to evaluate the remarkable association of two polymorphisms at 9p21 locus, rs1333049 and rs10757274, with CAD. Materials and Methods: This experimental study was conducted in Golestan, Aria Hospitals and Genetics Lab of Shahid Chamran University in the city of Ahvaz, Iran, in 2010- 2011. The collected blood samples belonging to 170 CAD patients (case group) and 100 healthy individuals (control group) were analyzed by tetra-primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) technique. The results were analyzed using software package used for statistical analysis (SPSS; SPSS Inc., USA) version 16. A value of p<0.05 and an odd ratio (OR) with 95% confidence intervals (CI) were considered significant. Results: The frequencies of CC, CG and GG genotypes for rs1333049 polymorphism in patients were 18.2, 65.3 and 16.5%, while in controls, the related values were 25, 67 and 8%, respectively. GG genotypes of rs1333049 polymorphism in CAD patients were more than control cases (OR: 0.354, 95%CI: 0.138-0.912, p=0.032). The frequencies of AA, AG and GG genotypes for rs10757274 in CAD patients were 8.2, 58.3 and 33.5%, while in controls, the related values were 35, 63 and 2%, respectively. GG Genotype in rs10757274 polymorphism in CAD patients was found more than control cases (OR: 0.014, 95% CI: 0.003 -0.065, p=0.0001). Conclusion: The rs1333049 polymorphism at 9p21 locus shows a weak association with CAD, whereas rs10757274 polymorphism reveals a significant association with CAD. These variants may help the identification of patients with increased risk for coronary artery disease.

111-120

Authors: Raziyeh Tahamtan , Ali Shabestani Monfared , Yasser Tahamtani , Alireza Tavassoli , Maasoomeh Akmali , Mohammad Amin Mosleh-Shirazi , Mohammad Mehdi Naghizadeh , Danial Ghasemi , Mojtaba Keshavarz , Gholam Hassan Haddadi

Objective: Free radicals generated by ionizing radiation attack various cellular components such as lipids. The lung is a very radiosensitive organ and its damage is a doselimiting factor in radiotherapy treatments. Melatonin (MLT), the major product of the pineal gland acts as a radioprotective agent. This study aims to investigate the radioprotective effects of MLT on malondialdehyde (MDA) levels and histopathological changes in irradiated lungs. Materials and Methods: In this experimental study, a total of 62 rats were divided into five groups. Group 1 received no MLT and radiation (unT), group 2 received oral MLT (oM), group 3 received oral MLT and their thoracic areas were irradiated with 18 Gy (oMR), group 4 received MLT by intraperitoneal (i.p.) injection and their thoracic areas were irradiated with 18 Gy (ipM-R), group 5 received only 18 Gy radiation in the thoracic area (R). Following radiotherapy, half of the animals in each group were sacrificed at 48 hours for evaluation of lipid peroxidation and early phase lung injuries. Other animals were sacrificed in the eighth week of the experiment for evaluation of the presence of late phase radiation induced lung injuries. Results: Pre-treatment of rats with either i.p injection (p<0.05) and oral administration of MLT (p<0.001) significantly reduced MDA levels in red blood cell (RBC) samples compared to the R group. Furthermore, i.p. injection of MLT decreased MDA levels in plasma and tissue (p<0.05). In the early phase of lung injury, both administration of MLT significantly increased lymphocyte (p<0.05) and macrophage frequency (p<0.001). MLT reduced the lung injury index in the lungs compared to the R group (p<0.05). Conclusion: The result of this study confirms the radioprotective effect of MLT on lipid peroxidation, and in both early and late phases of radiation induced lung injuries in an animal model.

129-136

Authors: Yousefreza Yousefniapasha , Gholamali Jorsaraei , Maryam Gholinezhadchari , Soleiman Mahjoub , Mahmoud Hajiahmadi , Mehrdad Farsi

Objective: Cigarette is a rich source of oxidants and reactive nitrogen species. Nitric oxide (NO) in high concentration has deleterious effects on human sperm function. Antioxidant defense system in seminal plasma protects spermatozoa from the attack of reactive oxygen metabolites. The purpose of this study was to determine the correlation between cigarette smoking with the NO levels and the total antioxidant capacity (TAC) of the seminal plasma in infertile smoker men and to compare severity of oxidative stress (OS) in them with fertile and infertile non-smoking men. Materials and Methods: In this cross sectional study, a total of 95 male participants attended the Infertility Clinic of the Fatehmeh-zahra Hospital in Babol, Mazandaran Province, Iran, between 2010 and 2011. They were divided into three groups: I. fertile non-smokers (F.ns; n=32), II. infertile non-smokers (IF.ns; n=30) and III. infertile smokers (IF.s; n=33) according to semen analysis World Health Organization guidelines (WHO, 2001) and smoking data. TAC concentration and NO levels of seminal plasma were measured using the ferric reducing ability of plasma (FRAP) method and the Griess reagent, respectively. Results: Standard sperm parameters were significantly higher in the fertile group than those in the infertile groups, but these differences between the IF.ns and IF.s were not statistically significant. The mean TAC in the seminal plasma was higher in the F.ns>IF.ns>IF.s, respectively, but these differences were not statistically significant among three groups (p= 0.096). In contrast, the mean NO level in the seminal plasma was lower in the F.ns

145-152

Authors: Ali Reza Rajabzadeh , Hossein Eimani , Homa Mohseni Koochesfahani , Abdol-Hossein Shahvardi , Rouhollah Fath

Objective: Ovarian and follicle transplantation may preserve fertility in young cancer survivors. In this study, we have transplanted preantral follicles using fibrin gel as a carrier and fibrin gel supplemented with platelet lysate (PL) as a rich source of angiogenic and growth factors. The purpose of this study was to evaluate the role of fibrin gel and PL in follicle transplantation. Materials and Methods: In this experimental study, ovaries were taken from 14-dayold Naval Medical Research Institute (NMRI) mice. Preantral follicles were dissected from the ovaries and encapsulated into fibrin gel supplemented with 5, 10, 15 or 20% PL, then transplanted back into the same donor mice. Fibrin gels supplemented with PL that contained preantral follicles were placed in a subcutaneous pocket in the back of the neck of the recipient, donor mouse (the same mouse that follicles were collected). After 14 days the grafts were processed and embedded in paraffin blocks, then serially sectioned for histological evaluation. We counted the follicles and classified them according to stage (preantral or antral). Data were presented as mean ± standard error of mean (SEM) and analysed by analysis of variance (ANOVA) and the Kruskal-Wallistest. Results: The mean percentage of recovered follicles encapsulated and transplanted in each group were 33.30 ± 2.47 (fibrin gel), 31.96 ± 1.90 (fibrin gel+5% PL), 34.02 ± 2.44 (fibrin gel+10% PL), 48.31 ± 2.06 (fibrin gel+15% PL) and 17.60 ± 2.79 (fibrin gel+20% PL). There was a significant increase in the recovery rate of grafted follicles with fibrin gel+15% PL (48.31%; p<0.001). The percentage of preantral follicles showed no significant difference in all groups (p<0.05). The percentage of antral follicles showed a significant decrease in follicles grafted with fibrin gel+20% PL when compared to the other groups (11.77%; p<0.005) but no significant difference was observed in the other groups. Conclusion: The use of PL in follicle transplantation can improve ovarian follicular survival rate.

159-169

Authors: Mina Jazaeri , Hosein Malekzadeh , Hamidreza Abdolsamadi , Loghman Rezaei-Soufi , Mohammad Samami

Although salivary alkaline phosphatase (ALP) can balance de- and remineralization processes of enamel, there is no evidence regarding its effects on the concentrations of calcium and phosphate in saliva. The present study aims to determine the relationship between salivary ALP activity and the concentrations of calcium and phosphate in saliva. In this cross-sectional study, we evaluated salivary markers in 120 males, ages 19 to 44 years. All participants provided 5 mL of unstimulated whole saliva and the level of enzyme activity as well as calcium and phosphate concentrations were measured using a colorimetric method. Data were gathered and analyzed by statistical package for social sciences (SPSS) 13.00 using Pearson correlation test. A p value of <0.05 was considered statistically significant. The mean age of participants in the present study was 32.95 ± 8.09 years. The mean pH of saliva was 6.65 ± 0.62. Salivary parameters included average ALP activity (5.04 ± 1.866 U/dL), calcium (4.77 ± 0.877 mg/dL) and phosphate (10.38 ± 2.301 mg/dL). Pearson correlation test showed no significant relationship between ALP activity and calcium and phosphate concentrations in saliva (p>0.05). According to the results of the present study, there was no significant relation between salivary ALP activity and calcium and phosphate concentrations in saliva. However, further research is highly recommended.

171-180

Authors: Soheila Manaflouyan Khajehmarjany , Seyed Ali Rahmani , Seyed Hadi Chavoshi , Ali Esfahani , Ali Akbar Movassaghpour Akbari

Chronic myeloid leukemia (CML) is a myeloproliferative disease. The cytogenetic hallmark of CML is Philadelphia (Ph) chromosome. This study aimed to diagnose suspected CML patients, to monitor CML patients under therapy using cytogenetic and fluorescence in situ hybridization (FISH) techniques to analyze their bone marrow (BM) and peripheral blood (PB) samples, and finally to compare their obtained results for both specimens. This study was conducted during one-year period (2012-2013). The participants were recruited from the Hematology and Oncology Clinic of Shahid Gazi (Emam Reza) Hospital of Tabriz University of Medical Sciences, Tabriz, East Azerbaijan Province, Iran. We analyzed 90 samples from 60 suspected CML patients (30 BM and 60 PB samples). All samples were analyzed using G-banding, 5 samples using dual fusion FISH (DF-FISH) probes, as well as 30 samples using both FISH and G-banding. Among the 90 analyzed samples of 60 patients, 25 (41.66%) were Ph+ using karyotyping, whereas five cases were not analyzable, so FISH was applied and the results confirmed that only two individuals were BCR-ABL+. In the comparison between 25 BM and 25 PB samples using karyotyping, 15 (60%) and 10 (40%) were ph+, respectively. The comparison of FISH and karyotyping on 30 samples showed that 9 (30%) and 8 (26.66%) were Ph+, respectively, and only 18.18% of Ph+ patients showed atypical patterns. In the comparison between BM-cytogenetic and PBinterphase- FISH (I-FISH), BM-cytogenetic was more reliable than PB-I-FISH in detecting Ph. Our data demonstrate that FISH analysis is a rapid, reliable and sensitive technique. The comparison between BM and PB showed that PB can not be replaced by BM, even in detecting by FISH.

392-405

Authors: Nahid Nasiri, Poopak Eftekhari-Yazdi

Assessment of embryo quality in order to choose the embryos that most likely result in pregnancy is the critical goal in assisted reproductive technologies (ART). The current trend in human in vitro fertilization/embryo transfer (IVF/ET) protocols is to decrease the rate of multiple pregnancies after multiple embryo transfer with maintaining the pregnancy rate at admissible levels (according to laboratory standards). Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection (ICSI) outcome has been frequently proposed in recent years. This article describes the current status of morphological embryo evaluation at different pre-implantation stages.

416-425

Authors: Sareh Asadi, Samaneh Dehghan, Maryam Hajikaram, Seyed Javad Mowla, Abolhassan Ahmadiani, Mohammad Javan

Objective: Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. Materials and Methods: In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results: Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). Conclusion: These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4.

440-447

Authors: Qin Yu, Lizhen Liu, Jie Lin, Yan Wang, Xiaobo Xuan, Ying Guo, Shaojun Hu

Objective: Transplantation of mesenchymal stem cells (MSCs) can promote functional recovery of the brain after hypoxic-ischemic brain damage (HIBD). However, the mechanism regulating MSC migration to a hypoxic-ischemic lesion is poorly understood. Interaction between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXC chemokine receptor 4 (CXCR4) is crucial for homing and migration of multiple stem cell types. In this study, we investigate the potential role of SDF-1α/CXCR4 axis in mediating MSC migration in an HIBD model. Materials and Methods: In this experimental study, we first established the animal model of HIBD using the neonatal rat. Bone marrow MSCs were cultured and labeled with 5-bromo-21-deoxyuridine (BrdU) after which 6×106 cells were intravenously injected into the rat. BrdU positive MSCs in the hippocampus were detected by immunohistochemical analyses. The expression of hypoxia-inducible factor-1α (HIF-1α) and SDF-1α in the hippocampus of hypoxic-ischemic rats was detected by Western blotting. To investigate the role of hypoxia and SDF-1α on migration of MSCs in vitro, MSCs isolated from normal rats were cultured in a hypoxic environment (PO2=1%). Migration of MSCs was detected by the transwell assay. The expression of CXCR4 was tested using Western blotting and flow cytometry. Results: BrdU-labeled MSCs were found in the rat brain, which suggested that transplanted MSCs migrated to the site of the hypoxic-ischemic brain tissue. HIF-1α and SDF- 1α significantly increased in the hippocampal formations of HIBD rats in a time-dependent manner. They peaked on day 7 and were stably expressed until day 21. Migration of MSCs in vitro was promoted by SDF-1α under hypoxia and inhibited by the CXCR4 inhibitor AMD3100. The expression of CXCR4 on MSCs was elevated by hypoxia stimulation as well as microdosage treatment of SDF-1α. Conclusion: This observation illustrates that SDF-1α/CXCR4 axis mediate the migration of MSCs to a hypoxic-ischemic brain lesion in a rat model.

456-465

Authors: Faezeh Alipour, Abbas Parham, Hossein Kazemi Mehrjerdi, Hesam Dehghani

Objective: Because of the therapeutic application of stem cells (SCs), isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs), have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs) from little amounts of fat tissue samples and determine some of their biological characteristics. Materials and Methods: In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3). The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials. Results: Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD) markers (CD29, CD44 and CD90) and not major histocompatibility complex II (MHC-II) and CD34 (hematopoietic marker). Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells. Conclusion: Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.

476-487

Authors: Ehsan Taghiabad, Sima Nasri, Saeed Shafieyan, Sasan Jalili Firoozinezhad, Nasser Aghdami

Objective: As a biological tissue material, amniotic membrane (AM) has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D) spongy scaffold made of the extracellular matrix (ECM) of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. Materials and Methods: In this experimental study, cellular components of human amniotic membrane (HAM) were removed with 0.03% (w/v) sodium dodecyl sulphate (SDS). Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs), collagen, and deoxyribonucleic acid (DNA). To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M) with pepsin (1 mg/ml). In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E) staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scafolds were cytocompatible. Results: Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml). Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg).With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% ± 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 μg/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 μm measured from scanning electron microscope (SEM) images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. Conclusion: The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation.

494-505

Authors: Mahmoud-Reza Rafiee,Afsaneh Malekzadeh Shafaroudi,Sara Rohban, Hamid Khayatzadeh,Hamid Reza Kalhor,Seyed Javad Mowla

Objective: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. Materials and Methods: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. Results: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Conclusion: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues.

514-527

Authors: Massoud Seifi,Ali Arayesh,Nafise Shamloo,Roya Hamedi

Objective: Orthodontically induced inflammatory root resorption (OIIRR) is considered to be an important sequel associated with orthodontic tooth movement (OTM). OTM after Socket preservation enhances the periodontal condition before orthodontic space closure. The purpose of this study is to investigate the histologic effects of NanoBone®, a new highly nonsintered porous nano-crystalline hydroxyapatite bone on root resorption following OTM. Materials and Methods: This experimental study was conducted on four male dogs. In each dog, four defects were created at the mesial aspects of the maxillary and mandibular first premolars. The defects were filled with NanoBone®. We used the NiTi closed coil for mesial movement of the first premolar tooth. When the experimental teeth moved approximately halfway into the defects, after two months, the animals were sacrificed and we harvested the area of interest. The first premolar root and adjacent tissues were histologically evaluated. The three-way ANOVA statistical test was used for comparison. Results: The mean root resorption in the synthetic bone substitute group was 22.87 ± 11.25×10-4 mm2 in the maxilla and 21.41 ± 11.25×10-4 mm2 in the mandible. Statistically, there was no significant difference compared to the control group (p>0.05). Conclusion: The use of a substitution graft in the nano particle has some positive effects in accessing healthy periodontal tissue following orthodontic procedures without significant influence on root resorption (RR). Histological evaluation in the present study showed osteoblastic activity and remodeling environment of nanoparticles in NanoBone®.

538-545

Authors: Elham Amirchaghmaghi,Abbas Rezaei,Ashraf Moini,Mohammad Ali Roghaei,Maryam Hafezi,Reza Aflatoonian

Objective: Unexplained recurrent spontaneous abortion (URSA) is one of the main complications of pregnancy which is usually defined as three or more consecutive pregnancy losses before the 20th week of gestation without a known cause. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and shown, along with its receptors (VEGFR1, 2), to play important roles in several physiologic processes including reproduction. The aim of the present study was to analyze gene expression of VEGF and VEGF receptors in endometrium of patients with a history of URSA compared with normal fertile women. In addition, serum VEGF concentration was assessed and compared between the two groups at the same time. Materials and Methods: In this case control study, endometrial and blood samples were obtained between day 19th and 24th of menstrual cycle (window of implantation) from 10 women with a history of URSA (case group) and 6 fertile women who had at least one successful pregnancy (control group). Expression of VEGF and VEGFRs was studied by reverse transcription- polymerase chain reaction (RT-PCR) and then quantified by real time PCR. Normalization of expression levels was done by comparison with beta-actin expression level as an internal control. Relative VEGF, VEGFR1 and VEGFR2 expression quantities were compared between the two groups. Enzyme linked immunosorbent assay (ELISA) was used for serum VEGF assay. Results: VEGF, VEGFR1 and VEGFR2 gene expression was detected in endometrial samples of both groups. The mean relative expression of VEGF gene was lower in the case group compared with control women, however, both VEGF receptors were expressed higher in endometrium of the case group. In addition, the serum level of VEGF was significantly higher in the case group compared with the controls. Conclusion: Alteration in gene expression of VEGF and its receptors in endometrium and changes of serum VEGF might play important roles in pathogenesis of unexplained RSA.

554-559

Authors: Issa Layali,Eisa Tahmasbpour,Manijeh Joulaei,Seyed Gholam Ali Jorsaraei,Parvin Farzanegi

Semen hyperviscosity (SHV) is one of the factors involved in deficiency in sperm function. This research aimed to evaluate seminal plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) levels in infertile patients with hyperviscous and non-hyperviscous semen samples to understand whether hyperviscous semen is associated with oxidative damage in infertile subjects. In this cross sectional study, 59 semen samples were provided by fertile (n=12) individuals as control, infertile patients with normal viscosity (n=25) and infertile patients with hyperviscosity (n=22). After semen parameters examination, semen viscosity was studied by glass pipettes. Seminal plasma TAC and MDA levels were measured by ferric reducing of antioxidant power (FRAP) and thiobarbituric acid reaction (TBAR) methods, respectively. A probability less than 0.05 was considered statistically significant throughout the article. The mean of sperm parameters including: counts, motility and normal morphology in patients with hyperviscosity were significantly lower than those in non-hyperviscosity patients (p<0.05, p<0.01 and p<0.001, respectively). The mean of seminal plasma TAC value in seminal plasma of non-hyperviscosity patients (1710.31 ± 458.67 μmol/l) was significantly (p<0.01) higher than that of hyperviscosity group (1230.25 ± 352 μmol/l). A trend toward a higher mean of seminal plasma MDA value was estimated for hyperviscous group compared with non-hyperviscous (1.01 ± 0.41 nmol/ml vs. 0.94 ± 0.28 nmol/l); however, it was nonsignificant. Hyperviscous semen impairs seminal plasma TAC which is eventually associated with sperm membrane lipid peroxidation.

564-567

Authors: Muhammad Irfan-Maqsood

Chromosome-centric human proteome project (C-HPP) is a recent initiative to rationalize and analyze gene-protein and protein-protein interactions in normal and disease conditions. This initiative is aimed to generate the proteomic atlas explaining the molecular architecture of the human body and was initiated in response to the hurdles identified during analyzing the human genome project (HGP). A need for the experimental observation of translated proteins was felt to analyze precisely what is going on in the cell. 25 countries around the world are participating in the C-HPP. This symposium report will introduce the Y-chromosome HPP which is undergoing in Iran by eminent molecular biologists of Royan Institute, Tehran and its collaborates.