Cell Journal (Yakhteh)

289-301

Authors: Zeinab Ghanavati, Mahmoud Orazizadeh, Vahid Bayati, Mohammad Reza Abbaspour, Layasadat Khorsandi, Esrafil Mansouri, Niloofar Neisi

Objective: The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic coculture technique. Materials and Methods: In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase- polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from coculture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). Results: The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups compared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. Conclusion: The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

310-321

Authors: Afsaneh Fazili, Soghra Gholami, Bagher Minaie Zangi, Ehsan Seyedjafari, Mahdi Gholami

Objective: This study examined the in vivo differentiation of mesenchymal stem cells (MSCs) into insulin producing cells (IPCs) on electrospun poly-L-lactide acid (PLLA) scaffolds coated with Matricaria chammomila L. (chamomile) oil. Materials and Methods: In this interventional, experimental study adipose MSCs (AMSCs) were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control) or PLLA scaffold coated with chamomile oil (experimental). A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs). Real-time polymerase chain reaction (PCR) determined the expressions of genes related to pancreatic endocrine development and function. Results: Fourier transform infrared spectroscopy (FTIR) results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm-1 for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001) compared to control group (0.063 ± 0.009, P≤0.05). Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation. Conclusion: The pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent) can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular mechanisms.

332-339

Authors: Maryam Naseroleslami, Kazem Parivar, Samideh Khoei, Nahid Aboutaleb

Objective: The label and detection of cells injected into target tissues is an area of focus for researchers. Iron oxide nanoparticles can be used to label cells as they have special characteristics. The purpose of this study is to examine the effects of iron oxide nanoparticles on human-derived amniotic membrane stem cell (hAMCs) survival and to investigate the magnetic properties of these nanoparticles with increased contrast in magnetic resonance imaging (MRI). Materials and Methods: In this experimental study, we initially isolated mesenchymal stem cells from amniotic membranes and analyzed them by flow cytometry. In addition, we synthesized superparamagnetic iron oxide nanoparticles (SPIONs) and characterized them by various methods. The SPIONs were incubated with hAMCs at concentrations of 25-800 μg/mL. The cytotoxicity of nanoparticles on hAMCs was measured by the MTT assay. Next, we evaluated the effectiveness of the magnetic nanoparticles as MRI contrast agents. Solutions of SPION were prepared in water at different iron concentrations for relaxivity measurements by a 1.5 Tesla clinical MRI instrument. Results: The isolated cells showed an adherent spindle shaped morphology. Polyethylene glycol (PEG)-coated SPIONs exhibited a spherical morphology. The average particle size was 20 nm and magnetic saturation was 60 emu/g. Data analysis showed no significant reduction in the percentage of viable cells (97.86 ± 0.41%) after 72 hours at the 125 μg/ml concentration compared with the control. The relaxometry results of this SPION showed a transverse relaxivity of 6.966 (μg/ml.s)-1 Conclusion: SPIONs coated with PEG used in this study at suitable concentrations had excellent labeling efficiency and biocompatibility for hAMCs.

346-352

Authors: Shokouhozaman Soleymanifard,Mohammad Taghi Bahreyni Toossi, Roghayeh Kamran Samani, Shokoufeh Mohebbi

Objective: Radiation effects induced in non-irradiated cells are termed radiation-induced bystander effects (RIBE). The present study intends to examine the RIBE response of QU-DB bystander cells to first, second and third radiation fractions and compare their cumulative outcome with an equal, single acute dose. Materials and Methods: This experimental study irradiated three groups of target cells for one, two and three times with 60Co gamma rays. One hour after irradiation, we transferred their culture media to non-irradiated (bystander) cells. We used the cytokinesis block micronucleus assay to evaluate RIBE response in the bystander cells. The numbers of micronuclei generated in bystander cells were determined. Results: RIBE response to single acute doses increased up to 4 Gy, then decreased, and finally at the 8 Gy dose disappeared. The second and third fractions induced RIBE in bystander cells, except when RIBE reached to the maximum level at the first fraction. We split the 4 Gy acute dose into two fractions, which decreased the RIBE response. However, fractionation of 6 Gy (into two fractions of 3 Gy or three fractions of 2 Gy) had no effect on RIBE response. When we split the 8 Gy acute dose into two fractions we observed RIBE, which had disappeared following the single 8 Gy dose. Conclusion: The impact of dose fractionation on RIBE induced in QU-DB cells depended on the RIBE dose-response relationship. Where RIBE increased proportionally with the dose, fractionation reduced the RIBE response. In contrast, at high doses where RIBE decreased proportionally with the dose, fractionation either did not change RIBE (at 6 Gy) or increased it (at 8 Gy).

362-370

Authors: Elham Ghanbari,Vahid Nejati, Mozafar Khazaei

Objective: This study aimed to evaluate the effects of royal jelly (RJ) on serum biochemical alterations and oxidative stress status in liver and pancreas of streptozotocin (STZ)- induced diabetic rats. Materials and Methods: In this experimental study, thirty two male Wistar rats were divided into the following four groups (n=8/group): i. Control (C), ii. Diabetic (D), iii. Royal jelly (R), and iv. Royal jelly-treated diabetic (D/R) groups. Diabetes was induced by single intraperitoneal (IP) injection of STZ (60 mg/kg). The RJ [100 mg/kg body weight (BW)] was administered orally for 42 days. Blood samples were used to determine serum levels of insulin, high density lipoprotein cholesterol (HDL-c), total protein (TP), albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and fasting blood glucose (FBG). Also, the antioxidant status was evaluated by determining the levels of malondialdehyde (MDA), catalase (CAT) and ferric reducing antioxidant power (FRAP) in liver and pancreas. Data were analyzed by one-way analysis of variance (ANOVA) with P<0.05 as the significant level. Results: STZ-induced diabetic rats showed a significant elevation in the serum levels of AST, ALT, ALP and FBG, whereas there was a significant decrease in serum levels of insulin, albumin, HDL-c and TP (P<0.05). Treatment of the diabetic rats with RJ restored the changes of the above parameters to their normal levels (P<0.05). In addition, RJ significantly improved reduced levels of FRAP and CAT as well as high MDA level in liver and pancreas (P<0.05). Conclusion: RJ improves oxidative damage induced by STZ in the liver and pancreas of rats; therefore, it can be considered as an effective and alternative treatment for diabetes.

381-388

Authors: Ali Zarei Mahmudabadi, Masoomeh Masoomi Karimi,Majid Bahabadi,Zahra Bagheri Hoseinabadi,Moslem JafariSani, Reza Ahmadi

Objective: Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles in angiogenesis of different developmental mechanisms such as wound healing, embryogenesis and diseases, including different types of cancer. VEGFR2 is associated with cell proliferation, migration, and vascular permeability of endothelial cells. Blocking VEGF and its receptors is suggested as a therapeutic approach to prevent tumor growth. In this study, we aim to block VEGF signaling via small interfering RNA (siRNA) inhibition of VEGFR2. Materials and Methods: In this experimental study, we used the RNA interference (RNAi) mechanism to suppress expression of the VEGFR2 gene. We conducted the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), Western blot, and flow cytometry analyses of VEGFR2 expression. Results: Real-time PCR and Western blot results showed that VEGFR2 expression significantly downregulated. This suppression was followed by inhibition of cell proliferation, reduction of viability, and induction of apoptosis in the cancer cells. Conclusion: These findings suggest that VEGFR2 has a role in cell proliferation and tumor growth. Accordingly, it is suggested that VEGFR2 can be a therapeutic target for controlling tumor growth and proliferation.

397-404

Authors: Somayeh Ahmadloo, Saeed Talebi,Mohammad Miryounesi, Parvin Pasalar, Mohammad Keramatipour

Objective: Methylmalonic acidura (MMA) is a rare autosomal recessive inborn error of metabolism. In this study we present a novel nucleotide change in the mutase (MUT) gene of two unrelated Iranian pedigrees and introduce the methods used for its functional analysis. Materials and Methods: Two probands with definite diagnosis of MMA and a common novel variant in the MUT were included in a descriptive study. Bioinformatic prediction of the splicing variant was done with different prediction servers. Reverse transcription- polymerase chain reaction (RT-PCR) was done for splicing analysis and the products were analyzed by sequencing. Results: The included index patients showed elevated levels of propionylcarnitine (C3). Urine organic acid analysis confirmed the diagnosis of MMA, and screening for mutations in the MUT revealed a novel C to G variation at the 3´ splice acceptor site in intron 12. In silico analysis suggested the change as a mutation in a conserved sequence. The splicing analysis showed that the C to G nucleotide change at position -3 in the acceptor splice site can lead to retention of the intron 12 sequence. Conclusion: This is the first report of a mutation at the position -3 in the MUT intron 12 (c.2125-3C>G). The results suggest that the identified variation can be associated with the typical clinical manifestations of MMA.

416-424

Authors: Hamze Badeli, Nader Shahrokhi, Mahdieosadat KhoshNazar, Majid Asadi-Shekaari, Mohammad Shabani,Hassan Eftekhar Vaghefi,Mohammad Khaksari,Mohsen Basiri

Objective: Following traumatic brain injury, disruption of blood-brain-barrier and consequent brain edema are critical events which might lead to increasing intracranial pressure (ICP), and nerve damage. The current study assessed the effects of aqueous date fruit extract (ADFE) on the aforementioned parameters. Materials and Methods: In this experimental study, diffused traumatic brain injury (TBI) was generated in adult male rats using Marmarou’s method. Experimental groups include two pre-treatment (oral ADFE, 4 and 8 mL/kg for 14 days), vehicle (distilled water, for 14 days) and sham groups. Brain edema and neuronal injury were measured 72 hours after TBI. Veterinary coma scale (VCS) and ICP were determined at -1, 4, 24, 48 and 72 hours after TBI. Differences among multiple groups were assessed using ANOVA. Turkey’s test was employed for the ANOVA post-hoc analysis. The criterion of statistical significance was sign at P<0.05. Results: Brain water content in ADFE-treated groups was decreased in comparison with the TBI+vehicle group. VCS at 24, 48 and 72 hours after TBI showed a significant increase in ADFE groups in comparison with the TBI+vehicle group. ICP at 24, 48 and 72 hours after TBI, was decreased in ADFE groups, compared to the TBI+vehicle. Brain edema, ICP and neuronal injury were also decreased in ADFE group, but VCS was increased following on TBI. Conclusion: ADFE pre-treatment demonstrated an efficient method for preventing traumatic brain deterioration and improving pathological parameters after TBI.

438-445

Authors: Majid Kamali-Dolat Abadi,Marziyeh Tavalaee, Abdolhossein Shahverdi,Mohammad Hossein Nasr-Esfahani

Objective: Globozoospermia is a rare type of teratozoospermia with incidence of 0.1% among infertile individuals. Phospholipase C zeta (PLCζ) and postacrosomal sheath WW domain binding protein (PAWP) are the main candidates in sperm taking responsibility for oocyte activation during fertilization. Therefore, we aimed to evaluate the expression of these two genes at RNA and protein levels in globozoospermic individuals and compare the results with fertile individuals. Materials and Methods: In this experimental study, semen samples of 21 infertile men with globozoospermia and 25 fertile men were collected. Expression of PLCζ and PAWP at RNA and protein levels were assessed and compared between two groups by quantitative real time polymerase chain reaction (qPCR) and Western blot, respectively. Results: Expression of both PLCζ and PAWP were significantly reduced at RNA and protein levels in infertile men with globozoospermia compared to fertile men. Conclusion: This is the first study that simultaneously assessing the respective factors in a large population of globozoospermia, suggested that intra-cytoplasmic sperm injection (ICSI) along with artificial oocyte activation may rescue failed fertilization in routine ICSI.

458-463

Authors: Mohsen Basiri, Majid Asadi-Shekaari,Masoud Ezzatabdipour, Arash Sarv Azad, Seyed Noureddin Nematollahimahani

Objective: Alcohol consumption is habitually accompanied by the use of other psychoactive substances, mostly tobacco. Nicotine and alcohol affect male accessory reproductive glands function. Most studies have been done on pathologic features of prostate, but there has been no systematic study on the seminal vesicle. Therefore, the aim of current study was to investigate the distribution of androgen receptor (AR) and estrogen receptors-beta (ER-β) immune reactivities following long-term treatment of alcohol, nicotine or a combination of both substances. Materials and Methods: In this experimental study, a total of 40 adult Wistar rats, nine weeks of age, were used. Animals were randomly divided into four groups, including: i. Control group receiving normal saline 0.09%, ii. Ethanol group receiving ethanol 20% (2 ml/kg, via gavage), iii. Nicotine group receiving nicotine (0.1 mg/kg, subcutaneous injection), and iv. Ethanol-nicotine group receiving simultaneous ethanol 20% (2 ml/kg) and nicotine (0.1 mg/kg) treatment. All treatment lasted for eight weeks. Prior to intracardiac perfusion, blood sample was collected from left ventricle. The seminal vesicles were isolated and processed for paraffin blocking. The sample tissues were then studied for distribution of AR and ER-β immunereactivities using immunohistochemical (IHC) staining method. One way analysis of variance (ANOVA) and Tukey’s test were performed for data analysis. A value of P<0.05 was considered significant. Results: Our results revealed that the lowest mean number of positive cells belonged to the animals of ethanol-nicotine group that was followed by the ethanol, nicotine, and control groups, respectively. However, there was no significant difference regarding serum testosterone level among experimental groups. Conclusion: It was concluded that combination of both ethanol and nicotine may be a crucial factor in the expression levels of AR and ER-β.

117-126

Authors: Saeid Saeidimehr, M.D., M.P.H.1, Ammar Ebrahimi, Ph.D.2, Najmaldin Saki, Ph.D.3, Parisa Goodarzi, M.Sc.4, Fakher Rahim, Ph.D.3

Ageing is a complex process and a broad spectrum of physical, psychological, and social changes over time. Accompanying diseases and disabilities, which can interfere with cancer treatment and recovery, occur in old ages. MicroRNAs (miRNAs) are a set of small non-coding RNAs, which have considerable roles in post-transcriptional regulation at gene expression level. In this review, we attempted to summarize the current knowledge of miRNAs functions in ageing, with mainly focuses on malignancies and all underlying genetic, molecular and epigenetics mechanisms. The evidences indicated the complex and dynamic nature of miRNA-based linkage of ageing and cancer at genomics and epigenomics levels which might be generally crucial for understanding the mechanisms of age-related cancer and ageing. Recently in the field of cancer and ageing, scientists claimed that uric acid can be used to regulate reactive oxygen species (ROS), leading to cancer and ageing prevention; these findings highlight the role of miRNA-based inhibition of the SLC2A9 antioxidant pathway in cancer, as a novel way to kill malignant cells, while a patient is fighting with cancer. Keywords: Ageing, Disability, Genomics, Longevity, MicroRNAs

135-148

Authors: Mohammad Mahdi Ochi, Ph.D.1, 2, Ghasem Amoabediny, Ph.D.2, 3*, Seyed Mahdi Rezayat, Ph.D.4, Azim Akbarzadeh, Ph.D.2, 5, Bahman Ebrahimi, Ph.D.1

Objective: This study aimed to evaluate a co-encapsulated pegylated nano-liposome system based on two herbal anti-tumor drugs, silibinin and glycyrrhizic acid, for delivery to a hepatocellular carcinoma (HCC) cell line (HepG2). Materials and Methods: In this experimental study, co-encapsulated nano-liposomes by the thin layer film hydration method with HEPES buffer and sonication at 60% amplitude. Liposomes that co-encapsulated silibinin and glycyrrhizic acid were prepared with a specified molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), and methoxy-polyethylene glycol 2000 (PEG2000)–derived distearoyl phosphatidylethanolamine (mPEG2000-DSPE). We used the MTT technique to assess cytotoxicity for various concentrations of co-encapsulated nano-liposomes, free silibinin (25% w/v) and glycyrrhizic acid (75% w/v) on HepG2 and fibroblast cell lines over a 48-hour period. Results: Formulation of pegylated nano-liposomes showed a narrow size distribution with an average diameter of 46.3 nm. The encapsulation efficiency (EE) for silibinin was 24.37%, whereas for glycyrrhizic acid it was 68.78%. Results of in vitro cytotoxicity showed significantly greater co-encapsulated nano-liposomes on the HepG2 cell line compared to the fibroblast cell line. The half maximal inhibitory concentration (IC50) for co-encapsulated pegylated nanoliposomal herbal drugs was 48.68 μg/ml and free silibinin with glycyrrhizic acid was 485.45 μg/ml on the HepG2 cell line. Conclusion: This in vitro study showed that nano-liposome encapsulation of silibinin with glycyrrhizic acid increased the biological activity of free drugs, increased the stability of silibinin, and synergized the therapeutic effect of silibinin with glycyrrhizic acid. The IC50 of the co-encapsulated nano-liposomes was lower than the combination of free silibinin and glycyrrhizic acid on the HepG2 cell line. Keywords: Encapsulation, Silibinin, Glycyrrhizic Acid, Hepatocellular Carcinoma

159-164

Authors: Mohammad Rafiee, M.Sc.1, Mohammad Reza Keramati, M.D.2*, Hosein Ayatollahi, M.D.2, Mohammad Hadi Sadeghian, M.D.2, Mohieddin Barzegar, M.Sc.1, Ali Asgharzadeh, M.Sc.1, Mohsen Alinejad, M.Sc.1

Objective: Signaling pathways such as extracellular regulated kinase/mitogen activated protein kinase (ERK/MAPK) have increased activity in leukemia. Ribosomal 6 kinase (RSK4) is a factor downstream of the MAPK/ERK pathway and an important tumor suppressor which inhibits ERK trafficking. Decrease in RSK4 expression has been reported in some malignancies, which leads to an increase in growth and proliferation and eventually poor prognosis. In this study we measured RSK4 expression rate in acute myeloid leukemia (AML). Materials and Methods: This cross-sectional study was undertaken in 2013-2014 at Ghaem Hospital in Mashhad, Iran, on 40 AML patients and 10 non-AML patients as the control group. The expression rate was measured by real-time polymerase change reaction (PCR) and employing the ΔΔCT method. Data were analyzed using Mann-Whitney and Spearman tests using SPSS (version 11.5). Results: Expression rate of RSK4 was significantly decreased in the AML group in comparison with the non-AML group (P<0.001). There was also a significant decrease in RSK4 expression in AML with t(15;17) in comparison to other translocations (P=0.004). Conclusion: We detected a down-regulation of RSK4 in AML patients. This may lead to an increase in the activity of the ERK/MPAK pathway and exacerbate leukemogenesis or the prognosis of the patients. Keywords: Extracellular Signal-Regulated Kinase, Mitogen Activated Protein Kinase, Ribosomal Protein S6 Kinase, Gene Expression, Acute Myeloid Leukemia

179-188

Authors: Abdolamir Allameh, Ph.D.1*, Maryam Jazayeri, Ph.D.1, 2, Maryam Adelipour, M.Sc.1

Objective: In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods: We induced human mesenchymal stem cells (MSCs) to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM) gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF), vascular endothelial growth factor (VEGF) receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID) mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC) staining. Results: Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E) staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion: The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis. Keywords: Mesenchymal Stem Cell, Endothelial Cells, Cell Transplantation, Differentiation

197-204

Authors: Abbas Kiani-Esfahani, M.Sc.1, Sedigheh Kazemi Sheykhshabani, M.Sc.2, Maryam Peymani, Ph.D.1, 3, Motahare-Sadat Hashemi, M.Sc.1, Kamran Ghaedi, Ph.D.1, 4*, Mohammad Hossein Nasr-Esfahani, Ph.D.1*

Objective: Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). Materials and Methods: In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Results: Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. Conclusion: ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions. Keywords: Annexin A1, Parkinson’s Disease, PC12 Cells, ROS

214-220

Authors: Khadijeh Falahzadeh, M.Sc.1, 2, Maryam Shahhoseini, Ph.D.1*, Parvaneh Afsharian, Ph.D.

Objective: Nuclear actin is involved in transcription regulation by recruitment of histone modifiers and chromatin remodelers to the regulatory regions of active genes. In recent years, further attention has been focused on the role of actin as a nuclear protein in transcriptional processes. In the current study, the epigenetic role of nuclear actin on transcription regulation of two stemness (OCT4 and NANOG) and two differentiation) NESTIN and PAX6) marker genes was evaluated in a human embryonal carcinoma cell line (NT2) before and after differentiation induction. Materials and Methods: In this experimental study, differentiation of embryonal cells was induced by retinoic acid (RA), and quantitative real-time polymerase chain reaction (PCR) was used to evaluate differential expression of marker genes before and 3 days after RAinduced differentiation. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR was then undertaken to monitor the incorporation of β-actin, as a functional component of RNA polymerase II, in the regulatory regions of marker genes. Results: Data showed significant change in nuclear actin incorporation into the promoter regions of NESTIN and PAX6 after RA-induction. Conclusion: We emphasize the dynamic functional role of nuclear actin in differentiation of embryonal cells and its role as a subunit of RNA polymerase II. Keywords: Nuclear Actin, NTera2/NT2, Retinoic Acid, Differentiation

229-236

Authors: Saeed Talebi, M.D., Ph.D.1, Mona Entezam, M.Sc.1, Neda Mohajer, M.Sc.1, Golnaz-Ensieh Kazemi-sefat, M.Sc.1, 2, Masoumeh Razipour, M.Sc.1, Somayeh Ahmadloo, M.Sc.1, Aria Setoodeh, M.D.3, Mohammad Keramatipour, M.D. Ph.D.1*

Objective: The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping. Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N’,N’-tetramethylrhodamine (HEX) and 6-carboxy- N,N,N’,N’-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software. Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001). Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems. Keywords: Microsatellite Repeats, Fluorescent Dyes, Capillary Electrophoresis, Diagnostic Error, Phenylalanine Hydroxylase

245-254

Authors: Sedigheh Ganji-Harsini, M.Sc., Mozafar Khazaei, Ph.D., Zahra Rashidi, M.Sc., Ali Ghanbari, Ph.D.*

Objective: Thymoquinone (TQ), as the main component of Nigella Sativa plant, shows anticancer properties. This study was aimed to evaluate the combined effect of TQ and Tamoxifen (TAM) on viability and apoptosis of human breast cancer cell lines. Materials and Methods: In this experimental study, estrogen positive MCF-7 and estrogen negative MDA-MB-231 human breast cancer cell lines were induced by TAM (2 μM) or different doses of TQ (50, 75, 100, 150 μM), individually or in combination. Cell viability and apoptosis were investigated by MTT assay and TdT-mediated deoxy-uracil nick end labeling (TUNEL) assay; Acridine orange (AO)/Ethidium bromide (EB) staining respectively. Data were analyzed by one way ANOVA and P<0.05 was considered significant. Results: In 24 hours treatment, TAM and all doses of TQ, solely or in combination, significantly reduced cell viability of both cell lines, except in MCF-7 cells treated with 50 μM TQ, and MDA-MB-231 cells treated with 50 or 75 μM TQ (P<0.01). After 48 hours treatment, cell viability of both cell lines was reduced in all treated groups (P<0.05). Remarkable apoptotic index was observed in combination treatment of MCF-7 or MDA-MB-231 cell lines with TAM and TQ (P<0.001). Conclusion: The synergistic effect of TQ and TAM on human breast cancer cell lines showed cell viability reduction as well as apoptosis induction, independent to estrogen. Keywords: Apoptosis, Breast Cancer, Necrosis, Tamoxifen, Thymoquinone.

262-20

Authors: AliReza Alizadeh, Ph.D.1, Zeinab Taleb, M.Sc.1, Bita Ebrahimi, Ph.D.2, Vahid Esmaeili, M.Sc.2, Abdolhossein Shaverdi, Ph.D.2*, Javad Nasr, Ph.D.1, Abolfazl Kheimeh, M.Sc.3, Reza Salman Yazdi, Ph.D.4

Objective: Although key roles for dietary vitamin E (VITE) and fatty acid (FA) in fertility have been confirmed, limited data are available on the effects of VITE alone, or a constant level of VITE supplemented by dietary omega-6 and omega-3 FAs in combination on male reproduction. Consequently in this paper, the effects of VITE, sunflower oil, fish oil and their combination on rat sperm were investigated. Materials and Methods: We divided 50 mature male Wistar rats into 5 groups (n=10) in a experimental completely randomized design for eight weeks: i. Control (CTR): standard diet; ii. Vitamin E diet (VITE): 2 times greater than recommendations; iii. Sunflower oil group (n-6) [gavaged with 0.5 ml/day/rat sunflower oil+VITE diet]; iv. Fish oil group (n-3): [gavaged with 0.5 ml/day/rat fish oil+VITE diet] and v. n-3+n-6 group [gavaged with 0.3 ml fish oil/day/rat+0.2 ml sunflower oil/day/rat+VITE diet]. The sperm parameters were measured by computer assisted semen analyzer (CASA). All data were analyzed with SPSS software. Results: Feed intake decreased in groups which were administered sunflower oil compared with the other groups (P<0.05). The groups which received only VITE or fish oil+VITE had a significantly higher concentration of sperm compared with the n- 6+n-3 and CTR group (P<0.05). VITE and n-3 showed significant improved progressive motility compared to the CTR group, whereas the n-6 and n-6+n-3 groups were in the middle (P<0.05). The highest sperm kinematic parameters were observed in the VITE only group. There was no strong correlation between sperm parameters and blood lipid profiles. Conclusion: Dietary VITE and fish oil+VITE can improve sperm quality. Our findings can be a focus for improvements in sperm quantity and motility in fertile animals using only dietary VITE. Keywords: Vitamin E, Fish Oil, Sunflower Oil, Rat, Sperm

281-287

Authors: Masoumeh Mousavinejad, M.Sc.1*, Peter W. Andrews, Ph.D.1, Elham Kargar Shoraki, M.Sc.2

Stem cells can be valuable model systems for drug discovery and modelling human diseases as well as to investigate cellular interactions and molecular events in the early stages of development. Controlling the differentiation of stem cells into specific germ layers provides a potential source of highly specialized cells for therapeutic applications. In recent years, finding individual properties of stem cells such as their ultimate self-renewal capacity and the generation of particular cell lines by differentiation under specific culture conditions underpins the development of regenerative therapies. These futures make stem cells a leading candidate to treat a wide range of diseases. Nevertheless, as with all novel treatments, safety issues are one of the barriers that should be overcome to guarantee the quality of a patient’s life after stem cell therapy. Many studies have pointed to a large gap in our knowledge about the therapeutic applications of these cells. This gap clearly shows the importance of biosafety concerns for the current status of cell-based therapies, even more than their therapeutic efficacy. Currently, scientists report that tumorigenicity and immunogenicity are the two most important associated cell-based therapy risks. In principle, intrinsic factors such as cell characteristics and extrinsic elements introduced by manufacturing of stem cells can result in tumor formation and immunological reactions after stem cell transplantation. Therapeutic research shows there are many biological questions regarding safety issues of stem cell clinical applications. Stem cell therapy is a rapidly advancing field that needs to focus more on finding a comprehensive technology for assessing risk. A variety of risk factors (from intrinsic to extrinsic) should be considered for safe clinical stem cell therapies. Keywords: Safety, Tumorigenicity, Immunogenicity

1-6

Authors: Sinha Abhishek, Suresh Palamadai Krishnan

The primary feature of the mammalian skin includes the hair follicle, inter-follicular epidermis and the sebaceous glands, all of which form pilo-sebaceous units. The epidermal protective layer undergoes an ordered/programmed process of proliferation and differentiation, ultimately culminating in the formation of a cornified envelope consisting of enucleated corneocytes. These terminally differentiated cells slough off in a cyclic manner and this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes. These genes, spanning 2 Mb region of human chromosome 1q21, play a crucial role in epidermal development, through various mechanisms. Each of these mechanisms employs a unique chromatin re-modelling factor or an epigenetic modifier. These factors act to regulate epidermal differentiation singly and/or in combination. Diseases like psoriasis and cancer exhibit aberrations in proliferation and differentiation through, in part, dysregulation in these epigenetic mechanisms. Knowledge of the existing mechanisms in the physiological and the aforesaid pathological contexts may not only facilitate drug development, it also can make refinements to the existing drug delivery systems. Keywords: Keratinocyte, Proliferation, Differentiation, Cornified Envelope, Drug Targeting

13-20

Authors: Mina Moayeri,, Hojatolah Saeidi, Mohammad Hossein Modarresi, Mehrdad Hashemi

Objective: Advanced maternal age (AMA) is an important factor in decreasing success of assisted reproductive technology by having a negative effect on the success rate of intra-cytoplasmic sperm injection (ICSI), particularly by increasing the rate of embryo aneuploidy. It has been suggested that the transfer of euploid embryos increases the implantation and pregnancy rates, and decreases the abortion rate. Preimplantation genetic screening (PGS) is a method for selection of euploid embryos. Past studies, however, have reported different results on the success of pregnancy after PGS in AMA. Investigating the pregnancy rate of ICSI with and without PGS in female partners over 35 years of age referred to infertility centers in Tehran. Materials and Methods: In this randomized controlled trial, 150 couples with the female partner over age of 35 were included. Fifty couples underwent PGS and the remaining were used as the control group. PGS was carried out using fluorescent in situ hybridization (FISH) for chromosomes 13, 18, 21, X and Y. Results of embryo transfer following PGS were evaluated and compared with those in the control group. Results: Implantation rates obtained in the PGS and control groups were 30 and 32% respectively and not significantly different (P>0.05). Conclusion: PGS for chromosomes 13, 18, 21, X and Y does not increase implantation rate in women over 35 years of age and therefore the regular use of PGS in AMA is not recommended. Keywords: Preimplantation Genetic Screening, Maternal Age, FISH Technique, Aneuploidies, ICSI

28-36

Authors: Ensieh M. Poursani, Bahram Mohammad Soltani, Seyed Javad Mowla,

Objective: The human OCT4 gene, the most important pluripotency marker, can generate at least three different transcripts (OCT4A, OCT4B, and OCT4B1) by alternative splicing. OCT4A is the main isoform responsible for the stemness property of embryonic stem (ES) cells. There also exist eight processed OCT4 pseudogenes in the human genome with high homology to the OCT4A, some of which are transcribed in various cancers. Recent conflicting reports on OCT4 expression in tumor cells and tissues emphasize the need to discriminate the expression of OCT4A from other variants as well as OCT4 pseudogenes. Materials and Methods: In this experimental study, DNA sequencing confirmed the authenticity of transcripts of OCT4 pseudogenes and their expression patterns were investigated in a panel of different human cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Results: Differential expression of OCT4 pseudogenes in various human cancer and pluripotent cell lines was observed. Moreover, the expression pattern of OCT4-pseudogene 3 (OCT4-pg3) followed that of OCT4A during neural differentiation of the pluripotent cell line of NTERA-2 (NT2). Although OCT4-pg3 was highly expressed in undifferentiated NT2 cells, its expression was rapidly down-regulated upon induction of neural differentiation. Analysis of protein expression of OCT4A, OCT4-pg1, OCT4-pg3, and OCT4-pg4 by Western blotting indicated that OCT4 pseudogenes cannot produce stable proteins. Consistent with a newly proposed competitive role of pseudogene microRNA docking sites, we detected miR-145 binding sites on all transcripts of OCT4 and OCT4 pseudogenes. Conclusion: Our study suggests a potential coding-independent function for OCT4 pseudogenes during differentiation or tumorigenesis. Keywords: OCT4 Pseudogenes, Stem Cell, Cancers, miR-145

46-51

Authors: Aram Rostami,Seyed Akbar Moosavi,Hassan Dianat Moghadam,Eftekhar Rajab Bolookat

Objective: Critical macromolecules such as DNA maybe damaged by free radicals that are generated from the interaction of ionizing radiation with biological systems. Melatonin and vitamin C have been shown to be direct free radical scavengers. The aim of this study was to investigate the in vivo/in vitro radioprotective effects of melatonin and vitamin C separately and combined against genotoxicity induced by 6 MV x-ray irradiation in human cultured blood lymphocytes. Materials and Methods: In this experimental study, fifteen volunteers were divided into three groups of melatonin, vitamin C and melatonin plus vitamin C treatment. Peripheral blood samples were collected from each group before, and 1, 2 and 3 hours after melatonin and vitamin C administration (separately and combined). The blood samples were then irradiated with 200 cGy of 6 MV x-ray. In order to characterize chromosomal aberrations, the lymphocyte samples were cultured with mitogenic stimulus on cytokinesisblocked binucleated cells. Results: The samples collected 1hour after melatonin and vitamin C (separately and combined) ingestion exhibited a significant decrease in the incidence of micronuclei compared with their control group (P<0.05). The maximum synergic protection and reduction in frequency of micronuclei (57%) was observed 1 hour after vitamin C and melatonin administration combined. Conclusion: We conclude that simultaneous administration of melatonin and vitamin C as radioprotector substances before irradiation may reduce genotoxicity caused by x-ray irradiation. Keywords: Radioprotective, Melatonin, Vitamin C, Lymphocyte, Micronucleus

62-73

Authors: Bent-al-hoda Movahedi Najafabadi, Mohammad Hussein Abnosi

Objective: Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). Materials and Methods: In this experimental study, BMSCs were extracted and expanded to the 3rd passage, then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 μg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. Results: Although 6 μg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. Conclusion: Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture. Keywords: Boric Acid, Mesenchymal Stem Cells, Morphology, Osteoblasts

83-88

Authors: Aslihan Saylan, Selcuk Duman

Objective: In 2014, enrolled 20 patients who applied to the Unit of Assisted Reproduction Techniques, Konya Necmettin Erbakan University. Based on the presence of hyaluronic acid (HA) in the oocyte-cumulus cell complex, sperm attached to HA in vivo were modeled in vitro. Available healthy sperm obtained in the swim-up procedure using HA were investigated. Materials and Methods: This observational cohort study, a routine analysis was conducted on the ejaculation samples obtained from 20 patients. We divided each sample into two groups and the swim-up method was applied. Human serum albumin (HSA, 0.5%) was added to samples from the first group. HA (10%) was added to samples from the second group. We determined the floating linear and non-linear sperm concentrations of both groups annexin V was used to determine the rate of apoptosis of these sperm. Results: Following swim-up, linear and non-linear sperm concentrations were higher in the group that contained HA compared to the group with HSA. However, there was a significantly higher apoptosis rate in the HSA group compared to the HA group. Conclusion: The addition of HA to the medium in the swim-up procedure positively affected sperm parameters. Thus, healthier sperm cells were obtained without DNA damage and with high motility. Keywords: Annexin V, Apoptosis, Hyaluronic Acid, Human

97-102

Authors: Mohammad Shokrzadeh, Hakimeh Abdi, Azin Asadollah-Pour, Fatemeh Shaki

Objective: Hyperglycemia, a common metabolic disorder in diabetes, can lead to oxidative damage. The use of antioxidants can benefit the control and prevention of diabetes side effects. This study aims to evaluate the effect of nanoceria particles, as an antioxidant, on glucose induced cytotoxicity, reactive oxygen species (ROS), lipid peroxidation (LPO) and glutathione (GSH) content in a human hepatocellular liver carcinoma cell line (HepG2) cell line. Materials and Methods: In this experimental study, we divided HepG2 cells into these groups: i. Cells treated with 5 mM D-glucose (control), ii. Cells treated with 45 mM Dmannitol+ 5 mM D-glucose (osmotic control), iii. Cells treated with 50 mM D-glucose (high glucose), and iv. Cells treated with 50 mM D-glucose+nanoceria. Cell viability, ROS formation, LPO and GSH were measured and analyzed statistically. Results: High glucose (50 mM) treatment caused significant cell death and increased oxidative stress markers in HepG2 cells. Interestingly, nanoceria at a concentration of 50 mM significantly decreased the high glucose-induced cytotoxicity, ROS formation and LPO. This concentration of nanoceria increased the GSH content in HepG2 cells (P<0.05). Conclusion: The antioxidant feature of nanoceria particles makes it an attractive candidate for attenuation of hyperglycemia oxidative damage in different organs. Keywords: Nanoceria, Hyperglycemia, Oxidative Stress, Cytotoxicity, HepG2

112-116

Authors: Ramesh Ahmadi, Mehdi Ahmadifar, Elham Safarpour, Nazila Vahidi-eyrisofla, Mehraneh Darab, Ali Mohammad Eini, AliReza Alizadeh

Levofloxacin is one of the Fluroquinoline antibiotic groups, which affect on controlling infections, especially in reproductive organs. It has therapeutic use in numerous countries, but little information exists on the effects of Levofloxacin on spermatogenesis when it is used for infectious treatment. The current study was designed to determine whether Levofloxacin influences testis tissue and spermatogenesis in rats. In this survey 50 male Wistar rats 6-8 weeks (250 ± 10 g) were used: normal salin as sham and control groups and 3 treatment groups (0.03, 0.06 and 0.08 mg Levofloxacin\kg body weight) during 60 days. The experimental groups were daily gavages. After 60 days, they were anesthetized with ether and testes were taken for histopathology studies, sperm parameters evaluation and several hormone concentrations. Although testosterone concentration was not affected by Levofloxacin levels, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentration significantly increased by Levofloxacin consumption in 0.03 and 0.06 mg Levofloxacin\kg body weight groups (P<0.01). Moreover, sperm concentration decreased linearly as Levofloxacin was increased (200, 192, 170, 128 and 75×106 sperm for control, sham, 0.03, 0.06 and 0.08 mg Levofloxacin\kg body weight, respectively, P<0.05). Testis tissue cuts in experimental group when the amount dosage of Levofloxacin increased cells solidarity to the primary and secondary spermatogonia. Adding Levofloxacin linearly reduced spermatocyte cells and amount of all cells in semenifer pipes tube (P<0.05). Levofloxacin as an antibiotic has histopathology effects on the spermatocyte cells, especially in high dose. Therefore, it might reduce fertility in male that requires further studies. Keywords: Levofloxacin, Spermatogenesis, Rat

583-592

Authors: Najmaldin Saki, Saeid Abroun, Masoud Soleimani, Maria Kavianpour, Mohammad Shahjahani, Javad Mohammadi-Asl, Saeideh Hajizamani

Today the regulatory role of microRNAs (miRs) is well characterized in many diverse cellular processes. MiR-based regulation is categorized under epigenetic regulatory mechanisms. These small non-coding RNAs participate in producing and maturing erythrocytes, expressing hematopoietic factors and regulating expression of globin genes by post-transcriptional gene silencing. The changes in expression of miRs (miR-144/-320/-451/-503) in thalassemic/sickle cells compared with normal erythrocytes may cause clinical severity. According to the suppressive effects of certain miRs (miR-15a/-16-1/-23a/-26b/-27a/-451) on a number of transcription factors [myeloblastosis oncogene (MYB), B-cell lymphoma 11A (BCL11A), GATA1, Krüppel-like factor 3 (KLF3) and specificity protein 1 (Sp1)] during β globin gene expression, It has been possible to increasing γ globin gene expression and fetal hemoglobin (HbF) production. Therefore, this strategy can be used as a novel therapy in infusing HbF and improving clinical complications of patients with hemoglobinopathies.

601-607

Authors: Elahe Motevaseli, Rosa Azam, Seyed Mohammad Akrami, Mohammadali Mazlomy, Mojtaba Saffari,Mohammad Hossein Modarressi, Maryam Daneshvar, Soudeh Ghafouri-Fard,

Objective: The aim of this study was to clarify the mechanism by which lactobacilli exert their cytotoxic effects on cervical cancer cells. In addition, we aimed to evaluate the effect of lactobacilli on the expression of human papilloma virus (HPV) oncogenes. Materials and Methods: In this experimental study, using quantitative real-time polymerase chain reaction (PCR), we analyzed the expression of CASP3 and three autophagy genes [ATG14, BECN1 and alpha 2 catalytic subunit of AMPK (PRKAA2)] along with HPV18 E6 and E7 genes in HeLa cells before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants. Results: The expression of CASP3 and autophagy genes in HeLa cells was decreased after treatment with lactobacilli culture supernatants. However, this decrease was not significant for PRKAA2 when compared with controls. In addition, expression of HPV E6 was significantly decreased after treatment with lactobacilli culture supernatants. Conclusion: Lactobacilli culture supernatants can decrease expression of ATG14 and BECN1 as well as the HPV E6 oncogene. It has been demonstrated that the main changes occurring during cervical carcinogenesis in cell machinery can be reversed by suppression of HPV oncogenes. Therefore, downregulation of HPV E6 by lactobacilli may have therapeutic potential for cervical cancer. As the role of autophagy in cancer is complicated, further work is required to clarify the link between downregulation of autophagy genes and antiproliferative effects exerted by lactobacilli. Keywords: HPV, Lactobacillus, Autophagy

617-628

Authors: Monireh Hajimoradi, Zuhair Mohammad Hassan, Marzieh Ebrahimi, Masoud Soleimani,Mahdieh Bakhshi, Javad Firouzi, Fazel Sahraneshin Samani

Objective: Gastric cancer (GC) is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcription- 3 (STAT3) signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell (ESCs) pluripotency. Here, we have investigated the activation status of STAT3 in GC stem-like cells (GCSLCs). Materials and Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, characterized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation. Results: Spheroid cells showed higher potential for spheroid formation than the parental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition (EMT) related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated (P<0.05), but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel (DTX) when compared with parental cells (P<0.05) according to the MTS assay. Although immunostaining and Western blotting showed expression of the STAT3 protein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 (p-STAT3) in spheroid structures relative to the parent cells according to flow cytometry analysis (P<0.05). Conclusion: The present findings point to STAT3 over activation in GCSLCs. Complementary experiments are required to extend the role of STAT3 in stemness features and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy. Keywords: Gastric Cancer, Cancer Stem Cells, Spheroid, STAT3, EMT

639-647

Authors: Zoleikha Golipoor, Fereshteh Mehraein, Fariba Zafari,Akram Alizadeh,Shima Ababzadeh,Maryam Baazm

Objective: Bone marrow (BM) is one of the major hematopoietic organs in postnatal life that consists of a heterogeneous population of stem cells which have been previously described. Recently, a rare population of stem cells that are called very small embryonic-like (VSEL) stem cells has been found in the BM. These cells express several developmental markers of pluripotent stem cells and can be mobilized into peripheral blood (PB) in response to tissue injury. In this study we have attempted to investigate the ability of these cells to migrate toward an injured spinal cord after transplantation through the tail vein in a rat model. Materials and Methods: In this experimental study, VSELs were isolated from total BM cells using a fluorescent activated cell sorting (FACS) system and sca1 and stage specific embryonic antigen (SSEA-1) antibodies. After isolation, VSELs were cultured for 7 days on C2C12 as the feeder layer. Then, VSELs were labeled with 1,1´-dioctadecyl-3,3,3´,3´- tetramethylindocarbocyanine perchlorate (DiI) and transplanted into the rat spinal cord injury (SCI) model via the tail vein. Finally, we sought to determine the presence of VSELs in the lesion site. Results: We isolated a high number of VSELs from the BM. After cultivation, the VSELs colonies were positive for SSEA-1, Oct4 and Sca1. At one month after transplantation, real-time polymerase chain reaction analysis confirmed a significantly increased expression level of Oct4 and SSEA-1 positive cells at the injury site. Conclusion: VSELs have the capability to migrate and localize in an injured spinal cord after transplantation. Keywords: Homing, Spinal Cord Injury, Migration

659-667

Authors: Marzieh Moghadas,Mohammad Amin Edalatmanesh,Reza Robati

Objective: The present study investigated the effects of gallic acid (GA) administration on trimethyltin chloride (TMT) induced anxiety, depression, and hippocampal neurodegeneration in rats. Materials and Methods: In this experimental study, the rats received intraperitoneal (i.p.) injections of TMT (8 mg/kg). The animals received either GA (50, 100 and 150 mg/kg) or saline as the vehicle for 14 consecutive days. We measured depression and anxiety levels of the rats by conducting the behavioral tail suspension (TST), elevatedplusmaze (EPM), and novelty suppressed feeding (NSF) tests. Histological analyses were then used to determine the cell densities of different hippocampal subdivisions. The data were analyzed with ANOVA and Tukey’s post hoc test. Results: GA administration ameliorated anxiety and depression in the behavioral tests. The cell densities in the CA1, CA2, CA3 and DG hippocampal subdivisionsfrom GA-treated rats were higher than saline treated rats. Conclusion: GA treatment against TMT-induced hippocampal degeneration altered cellular loss in the hippocampus and ameliorated the depression-anxiety state in rats. Keywords: Trimethyltin, Gallic Acid, Hippocampus, Cell Density

678-691

Authors: Zahra Zandieh,Fatemehsadat Amjadi, Mahnaz Ashrafi, Abbas Aflatoonian, Alireza Fazeli, Reza Aflatoonian

Objective: Toll like receptors (TLRs) are one of the main components of the innate immune system. It has been reported that expression of these receptors are altered in the female reproductive tract (FRT) during menstrual cycle. Here we used a fallopian tube epithelial cell line (OE-E6/E7) to evaluate the effect of two sex hormones in modulating TLR expression. Materials and Methods: In this experimental study, initially TLR gene expression in OEE6/ E7 cells was evaluated and compared with that of fallopian tube tissue using quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining. Thereafter, OE-E6/E7 cells were cultured with different concentrations of estradiol and progesterone, and combination of both. qRT-PCR was performed to reveal any changes in expression of TLR genes as a result of hormonal treatment. Results: TLR1-10 genes were expressed in human fallopian tube tissue. TLR1-6 genes and their respective proteins were expressed in the OE-E6/E7 cell line. Although estradiol and progesterone separately had no significant effect on TLR expression, their combined treatment altered the expression of TLRs in this cell line. Also, the pattern of TLR expression in preovulation (P), mensturation (M) and window of implantation (W) were the same for all TLRs with no significant differences between P, M and W groups. Conclusion: These data show the significant involvement of the combination of estradiol and progesterone in modulation of TLR gene expression in this human fallopian tube cell line. Further experiments may reveal the regulatory mechanism and signalling pathway behind the effect of sex hormones in modulating TLRs in the human FRT. Keywords: Estradiol, Progesterone, Fallopian Tube, Toll Like Receptors

701-710

Authors: Morteza Abouzaripour,Parichehr Pasbakhsh,Nader Atlasi,Abdol Hossein Shahverdi,Reza Mahmoudi,Iraj Ragerdi Kashani

Objective: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone marrow have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characterization with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochemistry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of PDX1 and GLUT2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion: Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical applications. Keywords: Stage-Specific Embryonic Antigen, Insulin-Secreting Cells, Cell Differentiation, Diabetes Mellitus

720-729

Authors: Layasadat Khorsandi, Sadegh Saremy, Ali Khodadadi, Fereshteh Dehbashi

Objective: To evaluate the effect of Exendine-4 (EX-4), a Glucagon-like peptide 1 (GLP-1) receptor agonist, on the differentiation of insulin-secreting cells (IPCs) from rat adipose-derived mesenchymal stem cells(ADMSCs). Materials and Methods: In this experimental study, ADMSCs were isolated from rat adipose tissue and exposed to induction media with or without EX-4. After induction, the existence of IPCs was confirmed by morphology analysis, expression pattern analysis of islet-specific genes (Pdx-1, Glut-2 and Insulin) and insulin synthesis and secretion. Results: IPCs induced in presence of EX-4 were morphologically similar to pancreatic islet-like cells. Expression of Pdx-1, Glut-2 and Insulin genes in EX-4 treated cells was significantly higher than the cells exposed to differentiation media without EX-4. Compared to EX-4 untreated ADMSCs, insulin release from EX-4 treated ADMSCs showed a nearly 2.5 fold (P<0.05) increase when exposed to a high glucose (25 mM) medium. The percentage of insulin positive cells in the EX-4 treated group was approximately 4-fold higher than in the EX-4 untreated ADMSCs. Conclusion: The present study has demonstrated that EX-4 enhances the differentiation of ADMSCs into IPCs. Improvement of this method may help the formation of an unlimited source of cells for transplantation. Keywords: Exendin-4, Differentiation, Insulin-Secreting Cells, Adipose Tissue, Mesenchymal Stem Cells

740-747

Authors: Maryam Sadat Fakhri-Bafghi, Seyedeh Farnaz Ghasemi-Niri, Sara Mostafalou, Mona Navaei-Nigjeh,Maryam Baeeri, Azadeh Mohammadirad, Mohammad Abdollahi

Objective: Organophosphorus (OP) compounds are used to control pests, however they can reach the food chain and enter the human body causing serious health problems by means of acetylcholinesterase (AChE) inhibition and oxidative stress (OS). Among the OPs, chlorpyrifos (CHP), malathion (MAL), and diazinon (DIA) are commonly used for commercial extermination purposes, in addition to veterinary practices, domestic, agriculture and public health applications. Two new recently registered medicines that contain selenium and other antioxidants, IMOD and angipars (ANG), have shown beneficial effects for OS related disorders. This study examines the effect of selenium-based medicines on toxicity of three common OP compounds in erythrocytes. Materials and Methods: In the present experimental study, we determined the efficacy of IMOD and ANG on OS induced by three mentioned OP pesticides in human erythrocytes in vitro. After dose-response studies, AChE, lipid peroxidation (LPO), total antioxidant power (TAP) and total thiol molecules (TTM) were measured in erythrocytes after exposure to OPs alone and in combined treatment with IMOD or ANG. Results: AChE activity, TAP and TTM reduced in erythrocytes exposed to CHP, MAL and DIA while they were restored in the presence of ANG and IMOD. ANG and IMOD reduced the OPs-induced elevation of LPO. Conclusion: The present study shows the positive effects of IMOD and ANG in reduction of OS and restoration of AChE inhibition induced by CHP, MAL and DIA in erythrocytes in vitro. Keywords: Human Erythrocyte, Organophosphorus, Oxidative Stress

755-755

Authors: Seyed Mohammad Javad Mortazavi, Hossein Mozdarani

We read with great interest an article by Shokri et al. entitled "Effects of Wi-Fi (2.45 GH z) exposure on apoptosis, sperm parameters and testicular histomorphometry in rats: a time course study" that is published in the latest issue of the Cell Journal (Vol.17, 2015: 322-331). In this article, Shokri et al. have presented their findings obtained in an experiment on an animal model. These researchers exposed rats to the 2.45 GHz radiation in a chamber with two Wi-Fi antennas on opposite walls of a box. The exposed animals in this study showed a decrease in sperm parameters. We have previously shown that exposure to electromagnetic fields generated by Wi-Fi routers or mobile phone jammers can adversely affect the sperm quality (1-3). The paper published by Shokri et al. is seriously flawed. The first major shortcoming of this paper is its exposure geometry. The authors stated that their exposure system was "a chamber (180 cm×80 cm×70 cm), designed for whole-body exposure of free-moving rats to a Wi-Fi signal. Two Wi-Fi antennas (NanoStation Loco M2, 2.45 GHz, 8.5 dBi, Ubiquiti Networks, Inc. USA) were placed at the center of two sides of the chamber". It should be noted that in this case, the power density can be calculated using the below equation: S=P•G/4πR2 Where S=Power density P=Power input to antenna G=Antenna gain In this light, the geometry used in the study of Shokri et al. makes a very inhomogeneous distribution of power densities. The second shortcoming comes from this point that the authors claimed that their study was performed on a basis that could not affect the hormonal balance "A previous study applied a restrainer to fix space between antenna and rat. Since it was a stressful condition that could probably affect hormonal balance of animals, we tried to assess the effect of radiation on the free moving animals". However, these authors only had a control group and did not use a sham-exposed group to control the animals’s stress and its subsequent hormonal changes. Furthermore, another shortcoming comes from this point that "NanoStation Loco M2" is not a standard Wi-Fi router. As manufacturer reports this device is a compact outdoor communication unit that can be used for devices such as cameras "NanoStation Loco M2 is a compact outdoor unit which includes 2×8 dBi antenna (MIMO) for the 2.4 GHz band”. Therefore, it is misleading to claim that in this study the effects of Wi-Fi exposure on apoptosis are investigated and the title of this paper is indeed incorrect "Effects of Wi-Fi (2.45 GH z) exposure on apoptosis, sperm parameters and testicular histomorphometry in rats”. We hope that these comments are helpful to make more reliable results in the future.