Cell Journal (Yakhteh)

302-311

Authors: Suresh Palamadai krishnan

Breast cancer (BC) is a widely prevalent form of neoplasia in women with fairly alarming mortality statistics. This aspect may be attributed, in part, to the current spatial and temporal heterogeneity-based limitations in therapies with possible recurrence of this tumour at primary and/or secondary sites. Such an extensive phenotypic heterogeneity in breast cancer is unlikely to be adequately or completely comprehended by an immuno-histopathology-based classification alone. This finding has warranted research and development in the area of microarray-based methods (i.e. transcriptomic and proteomic chips) for an improved molecular classification of this complex and heterogeneous tumour. Further, since epigenetics can also be an important determinant in terms of diagnosis, prognosis and therapy, this review provides an insight into the molecular portrait of BC in genetic and epigenetic terms. Specifically, the roles of characteristic DNA and histone-based modifications as well as mi-RNA- based alterations have been discussed with specific examples. Also, their involvement in epithelial mesenchymal transition (EMT) processes in cancer stem cells (CSCs) has been outlined. Last but not least, the salient aspects and the advantages of ex vivo/in vitro 3D model systems in recapitulating several aspects of BC tumour (particularly the architecture as well as the apico-basal polarity) are mentioned. This review hopes to provide not only an improved and updated understanding of the epigenetics of breast cancer, but to also elaborate on tumour model development/refinement, biomarker evaluation, drug resistance and test of individual drugs or drug combinations and drug delivery systems.

318-325

Authors: Reza Ranjbaran, Mahin Nikogoftar Zarif, Sedigheh Sharifzadeh, Habibollah Golafshan, Ali Akbar Pourfathollah

Objective Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. Conclusion Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

333-339

Authors: Fatemeh Bahreini, Massoud Houshmand, Mohammad Hossein Modaresi, Seyed Mohammad Akrami

Objective Pompe disease (PD) is a progressive neuromuscular disorder that is caused by glucosidase acid alpha (GAA) deleterious mutations. Mitochondrial involvement is an important contributor to neuromuscular diseases. In this study the sequence of MT-ATP 6/8 and Cytochrome C oxidase I/II genes along with the expression levels of the former genes were compared in classic and non-classic patients. Materials and Methods In this case-control study, the sequence of MT-ATP 6/8 and Cytochrome C oxidase was analyzed by polymerase chain reaction (PCR)-Sanger sequencing and expression of MT-ATP genes were quantified by real time-PCR (RT-PCR) in 28 Pompe patients. The results were then compared with 100 controls. All sequences were compared with the revised Cambridge reference sequence as reference. Results Screening of MT-ATP6/8 resulted in the identification of three novel variants, namely T9117A, A8456C and A8524C. There was a significant decrease in MT-ATP6 expression between classic (i.e. adult) and control groups (P=0.030). Additionally, the MT-ATP8 expression was significantly decreased in classic (P=0.004) and non-classic (i.e. infant) patients (P=0.013). In total, 22 variants were observed in Cytochrome C oxidase, five of which were non- synonymous, one leading to a stop codon and another (C9227G) being a novel heteroplasmic variant. The A8302G in the lysine tRNA gene was found in two brothers in a pedigree, while a T7572C variant in the aspartate tRNA gene was observed in two brothers in another pedigree. Conclusion The extent of mitochondrial involvement in the classic group was more significant than in the non-classic form. Beside GAA deleterious mutations, it seems that mtDNA variants have a secondary effect on PD. Understanding, the role of mitochondria in the pathogenesis of Pompe may potentially be helpful in developing new therapeutic strategies.

348-354

Authors: Malihe Nakhaeifard, Maryam Haji ghasem kashani, Iran Goudarzi, Arezou Rezaei

Objective Adipose derived stem cells (ASCs) secrete numerous neurotrophic factors and cytokines in conditioned medium (CM), which protect neurons by its antioxidative and trophic effects. This research assesses the neuroprotective effect of ASC- CM on neurotrophins genes expressions and tyrosine hydroxylase positive (TH+) cell density in male Wistar rats lesioned by 6-hydroxydopamine (6-OHDA). Materials and Methods In this experimental study, the groups consisted of lesioned and sham rats with unilateral injections of 20 µg of 6-OHDA neurotoxin and phosphate buffered saline (PBS) into the striatum, respectively. Another groups received intravenous injections of 3×106 cells (ASCs group), 500 µl of CM (ASC-CM group) or medium [α-minimal essential medium (α-MEM) group)]. All rats underwent evaluations with the rotarod and apomorphine-induced rotation tests at 2, 4, 6, and 8 weeks post-injection. At 8 weeks we sacrificed some of the animals for real-time polymerase chain reaction (PCR) analysis, and evaluation of TH+cell counts. Results We observed a significant decrease in contralateral turns to the lesions in the ASCs and ASC-CM groups compared to the neurotoxin lesioned or α-MEM groups at 8 weeks post transplantation. Cell and CM- injected rats showed a significant increase of staying on the rotarod compared to the lesion or α-MEM groups. Cell and CM-treated rats showed significant increases in the NGF and NT3 genes, respectively, compared with the lesion group. Both treated groups showed significant increases in BDNF gene expression and TH+ cell density. Conclusion The results suggested that ASCs and ASC-CM protected dopaminergic neurons through the expressions of neurotrophin genes.

361-368

Authors: Azam Samadian, Mahdi Hesaraki, Sepideh Mollamohamadi, Behrouz Asgari, Mehdi Totonchi, Hossein Baharvand

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. Conclusion Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.

377-387

Authors: Zahra Azhdari Tafti, Mehdi Mahmoodi, Mohamas Reza Hajizadeh, Vahid Ezzatizadeh, Hossein Baharvand, Massoud Vosough, Abbas Piryae

Objective Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes. Materials and Methods In this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William’s) medium supplemented with 4% N-CM or H-CM. Untreated William’s and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells. Results We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063). Conclusion The enrichment of William’s basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.

396-402

Authors: Nassim Ghorbanmehr, Mojdeh Salehnia, Mahboobeh Amoushahi

Objective The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite (SS) on oocyte mitochondrial DNA (mtDNA) copy number and reactive oxygen species (ROS) levels. Materials and Methods In this experimental study germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were cultured and matured in the presence and absence of SS. Then in vivo and in vitro matured (IVM) oocytes were subjected to mitochondria staining by MitoTracker green, ROS analysis, and mtDNA copy number determination using absolute real-time polymerase chain reaction (PCR). Results The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group (79.25%) compared to the control group (72.46%, P<0.05). The intensity of mitochondrial staining was not different among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some aggregation pattern. The in vivo obtained MII oocytes had lower ROS levels and higher mtDNA copy numbers than IVM-MII oocytes (P<0.05). The SS supplemented group had significantly lower ROS levels and higher mtDNA copy numbers than the non-treated group (P<0.05). Conclusion SS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence.

412-421

Authors: Mozafar Khazaei, Mona Pazhouhi, Saber Khazaei

Objective Glioblastoma multiforme is the most malignant form of brain tumors. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. Here we have investigated the effects of T. pratense extract on glioblastoma multiforme cell line (U87MG). Materials and Methods In this experimental study the effect of T. pratense extract on cell viability was investigated using trypan blue staining, MTT assay, and lactate dehydrogenase activity measurement. Apoptosis and autophagy cell death were detected by fluorescent staining. Nitric oxide (No) production was measured using Griess reaction. Expression levels of some apoptotic and autophagic-related genes were detected using real-time polymerase chain reaction (PCR). The combination effects of T. pratense extract and temozolomide (TMZ) were evaluated by calculating the combination index and dose reduction index values. Results After treatment with T. pratense extract, the cell viability was significantly reduced in a time- and dose- dependent manner (P<0.05). Apoptosis and autophagy of U87MG cells were significantly increased (P<0.05). Also, T. pratense extract significantly decreased NO production (P<0.05) by U87MG cells. Combination of TMZ and T. pratense extract had a synergistic cytotoxic effect. Conclusion T. pratense showed anti-cancer properties via induction of apoptosis and autophagy cell death.

427-434

Authors: neda Ranjbar Kohan, Saeed Nazifi, Mohamad Reza Tabandeh, Maryam Ansari Lari

Objective L-carnitine (LC) has been shown to protect cardiac metabolism in diabetes patients with cardiovascular diseases (CVDs). Apelin, a newly discovered adipocytokines, is an important regulator of cardiac muscle function; however, the role of the level of expression of Apelin axis in improvement of cardiac function by LC in diabetic patients, is not clear. In the present study, obese insulin-resistant rats were used to determine the effect of LC, when given orally with a high-calorie diet, on Apelin and Apelin receptor (Apj) expression in cardiac muscle. Materials and Methods In this experimental study, rats were fed with high-fat/high-carbohydrate diet for five weeks and subsequently were injected with streptozotocin 30 mg/kg for induction of obesity and insulin resistance. After confirming the induction of diabetes (serum glucose above 7.5 mmol/L), the animals received LC 300 mg/kg in drinking water for 28 days. On days 0, 14 and 28 after treatment, cardiac Apelin and Apj gene expression was evaluated by real time polymerase chain reaction (PCR) analysis. Serum levels of insulin, Apelin, glucose, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and the homeostasis model assessment of insulin resistance (HOMA-IR) were also measured using commercial kits. Results Cardiac Apelin and Apj expression and serum Apelin were increased in obese rats, while LC supplementation decreased the serum levels of Apelin and down-regulated Apelin and Apj expression in cardiac muscle. These changes were associated with reduced insulin resistance markers and serum inflammatory factors and improved lipid profile. Conclusion We concluded that LC supplementation could attenuate the over-expression of Apelin axis in heart of diabetic rats, a novel mechanism by which LC improves cardiovascular complications in diabetic patients.

443-448

Authors: Mahdi Lotfipanah, Fereydoon Azadeh, Mehdi Totonchi, Reza Omani-Samani

Objective Stem cells that have unlimited proliferation potential as well as differentiation potency are considered to be a promising future treatment method for incurable diseases. The aim of the present study is to evaluate the future trend of stem cell researches from researchers’ viewpoints. Materials and Methods This was a cross-sectional descriptive study on researchers involved in stem cell research at Royan Institute. We designed a questionnaire using a qualitative study based on expert opinion and a literature review. Content validity was performed using three rounds of the Delphi method with experts. Face validity was undertaken by a Persian literature expert and a graphics designer. The questionnaire was distributed among 150 researchers involved in stem cell studies in Royan Institute biology laboratories. Results We collected 138 completed questionnaires. The mean age of participants was 31.13 ± 5.8 years; most (60.9%) were females. Participants (76.1%) considered the budget to be the most important issue in stem cell research, 79.7% needed financial support from the government, and 77.5% felt that charities could contribute substantially to stem cell research. A total of 90.6% of participants stated that stem cells should lead to commercial usage which could support future researches (86.2%). The aim of stem cell research was stipulated as increasing health status of the society according to 92.8% of the participants. At present, among cell types, importance was attached to cord blood and adult stem cells. Researchers emphasized the importance of mesenchymal stem cells (MSCs) rather than hematopoietic stem cells (HSCs, 57.73%). The prime priorities were given to cancer so that stem cell research could be directed to sphere stem cell research whereas the least preference was given to skin research. Conclusion Regenerative medicine is considered the future of stem cell research with emphasis on application of these cells, especially in cancer treatment.

138-149

Authors: Hamid Chobineh, Mahsa Kazemi, Mohammad Ali Sadighi Gilani, Tahereh Heydari, Saeed Shokri, Mahshid Bazrafkan, Gholamreza Hassanzadeh

Objective: This study evaluated the effects of exogenous testosterone molecule-1 (CADM1) pathological defect during early and chronic periods of spinal cord injury (SCI). Materials and Methods: In this experimental study, testosterone was administered immediately or after one week of SCI induction. Along with quantification of CADM1 gene expression and its immunoreactivity, we evaluated sperm parameters and serum testosterone level post-SCI. Results: Different grades of abnormalities in sperm parameters and testis architecture were observed along with significant reductions in the level of CADM1 expression and its immunoreactivity in the seminiferous tubules of both acute and chronic SCI groups. Exogenous testosterone, by compensating the serum testosterone level. reduced the percentage of apoptotic and both short head and abnormal sperm froms in the caudal epididymis. Importantly, the beneficial effects of immediate administration of testosterone were prominent. Increases in the level of CADM1 transcription and its immunoreactivity in the testis of SCI mice treated with testosterone were accompanied by improvement of sperm motility as well as testicular Johnsen’s and Miller’s criteria. Conclusion: Since immediate testosterone treatment improved the immunoreactivity and transcription level of CADM1, the observed beneficial effect of exogenouse testosterone can be attributed to its effect on CADM1 dynamics.

157-167

Authors: Mohamadreza Afarinesh, Gila Behzadi

Objective: The aim of the present study is to investigate the effects of chronic whisker deprivation on possible alterations to the development of nitrergic neurons in the whisker part of the somatosensory (wS1) and motor (wM1) cortices in offspring with congenital hypothyroidism (CH). Materials and Methods: In the experimental study, CH was induced by adding propylthiouracil to the rats drinking water from embryonic day 16 to postnatal day (PND) 60. In whisker-deprived (WD) pups, all the whiskers were trimmed from PND 1 to 60. Nitrergic interneurons in the wS1/M1 cortices were detected by NADPH-diaphorase histochemistry staining technique in the control (Ctl), Ctl+WD, Hypo and Hypo+WD groups. Results: In both wS1 and wM1 cortices the number of nitrergic neurons was significantly reduced in the Hypo and Hypo+WD groups compared to Ctl and Ctl+WD groups, respectively (P<0.05) while bilateral whisker deprivation had no remarkable effect. The mean soma diameter size of NADPH-d labeled neurons in the Ctl+WD and Hypo+WD groups was decreased compared to the Ctl and Hypo groups, respectively. A similar patterns of decreased NADPH-d labeled neurons in the wS1/M1 cortices occur in the processes of nitrergic neurons in both congenital hypothyroidism and whisker deprivation. Conclusion: Our results suggest that both congenital hypothyroidism and whisker deprivation may disturb normal development of the wS1 and wM1 cortical circuits in which nitrergic neurons are involved.

177-182

Authors: Azadeh Seyedjoodaki, Fereshteh Alsahebfosoul, Nahid Eskandari, Vahid Shaygannejad, Mansoor Salehi, Mohammad Kazemi, Mostafa Manian, Omid Mirmosayyeb, Mohmmad Taghi Kardi

Inflammation of the immune system and the central nervous system has been known as an important predisposing factor for Parkinson’s disease (PD). Increased expression of OX40 protein (CD134) is a known factor for increased inflammation and initiation of NF-kappa-B signaling pathway in different diseases. We aimed to investigate the expression of OX40 at the transcript and serum protein levels. Materials and Methods: Twenty individuals with PD and 20 healthy individuals, as controls, were enrolled in this casecontrol study. Expression of OX40 at the transcript level and serum protein levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays respectively. Results: The mean expression level of OX40 was increased in patients but not at a significant level (P>0.05). Consistently, the mean serum concentration of OX40 showed a mild, but non-significant, increase in the patients (P>0.05). Conclusion: We conclude that OX40 expression at either the transcript or protein level has no diagnostic utility in asymptomatic PD. This shows the need for clinical, cellular and interventional research to detect new robust biomarkers.

188-194

Authors: Milad Ahani-Nahayati, Saeed Solali, Karim Shams Asanjan, Ali Akbar Movassaghpour Akbari, Mehdi Talebi, Milad Zadi Heydarabad, Sina Baharaghdam, Majid Farshdousti Hagh

Objective: DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes. Materials and Methods: In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes’ promoter regions. Results: The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2 and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes. Conclusion: Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies.

204-210

Authors: Hossein Pashaiefar, Marjan Yaghmaie, Javad Tavakkoly-Bazzaz, Seyed Hamidollah Ghaffari, Kamran Alimoghaddam, Pantea Izadi, Ardeshir Ghavamzadeh

Objective: Chromosomal translocations are among the most common mutational events in cancer development, especially in hematologic malignancies. However, the precise molecular mechanism of these events is still not clear. It has been recently shown that alternative non-homologous end-joining (alt-NHEJ), a newly described pathway for double-stranded DNA break repair, mediates the formation of chromosomal translocations. Here, we examined the expression levels of the main components of alt-NHEJ (PARP1 and LIG3) in acute myeloid leukemia (AML) patients and assessed their potential correlation with the formation of chromosomal translocations. Materials and Methods: This experimental study used reverse transcription-quantitative polymerase chain reaction (RTqPCR) to quantify the expression levels of PARP1 and LIG3 at the transcript level in AML patients (n=78) and healthy individuals (n=19). Results: PARP1 was the only gene overexpressed in the AML group when compared with healthy individuals (P=0.0004), especially in the poor prognosis sub-group. Both genes were, however, found to be up-regulated in AML patients with chromosomal translocations (P=0.04 and 0.0004 respectively). Moreover, patients with one isolated translocation showed an over-expression of only LIG3 (P=0.005), whereas those with two or more translocations over-expressed both LIG3 (P=0.002) and PARP1 (P=0.02). Conclusion: The significant correlations observed between PARP1 and LIG3 expression and the rate of chromosomal translocations in AML patients provides a molecular context for further studies to investigate the causality of this association.

220-230

Authors: Sara Eslamizadeh, Mansour Heidari, Shahram Agah, Ebrahim Faghihloo, Hossein Ghazi, Alireza Mirzaei, Abolfazl Akbari

Objective: Colorectal cancer (CRC) is one of the most common cancers and a major cause of cancer-related death worldwide. The early diagnosis of colorectal tumors is one of the most important challenges in cancer management. MicroRNAs (miRNAs) have provided new insight into CRC development and have been suggested as reliable and stable biomarkers for diagnosis and prognosis. The aim of this study was to analyze the differential expression of miRNAs at different stages of CRC searching for possible correlation with clinicopathological features to examine their potential value as diagnostic biomarkers. Materials and Methods: In this case-control study, plasma and matched tissue samples were collected from 74 CRC patients at stage II-IV as well as blood samples from 32 healthy controls. After exhaustive study of the current literature, eight miRNAs including miR-200c, 20a, 21, 31,135b, 133b,145 and let-7g were selected. The expression level of the miRNAs was assayed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis, including t test , Mann-Whitney U, Kruskall-Wallis tests and receiver operating characteristic (ROC) curve was applied, where needed. Results: Significantly elevated levels of miR-21, miR-31, miR-20a, miR-135b, and decreased levels of miR- 200c, miR-145 and let-7 g were detected in both plasma and matched tissue samples compared to the healthy group (P<0.05). However, no significant differences were observed in the expression level of plasma and tissue miR-133b (P>0.05). ROC for tissue miRNAs showed an area under the ROC curve (AUC) of 0.98 and P<0.001 for miR-21, 0.91 and P<0.001 for miR-135b, 0.91 and P<0.001 for miR-31, and 0.92 and P<0.001 for miR-20a. Conclusion: Our results indicate that the expression levels of microRNAs are systematically altered in CRC tissue and plasma. In conclusion, detection of miR-21, miR-135b, miR-31 and miR-20a levels in the tissue might be helpful to illuminate the molecular mechanisms underlying CRC carcinogenesis and serve as tumor-associated biomarkers for diagnosis.

244-249

Authors: Somayeh Keshavarzi, Mohammad Salehi, Fttaneh Farifteh-Nobijari, Taher Hosseini, Sara Hosseini, Alaleh Ghazifard, Marefat Ghaffari Novin, Vahid Fallah-Omrani, Mohsen Nourozian, Ahmad Hosseini

Objective: We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione (GSH) and reactive oxygen species (ROS) levels, as well as histone H3 lysine 9 (H3K9), and H4 lysine 12 (H4K12) acetylation when added to oocytes culture medium. Materials and Methods: In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes (COCs) from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin (10-9 and 10-6 M) and without melatonin [control group treated with dimethyl sulfoxide (DMSO)] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane (MCB) and 2-7-dichlorodihydrofluorescein diacetate (H2DCFDA) staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12. Results: The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group (P<0.05). Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group (P<0.05). Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group (P<0.05). Conclusion: Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation (IVM).

259-266

Authors: Abdolkhaleg Deezagi, Samira Shomali

Objective: Tissue engineering today uses factors that can induce differentiation of mesenchymal stem cells (MSCs) into other cell types. However, the problem of angiogenesis in this differentiated tissue remains an unresolved area of research interest. The aim of this study was to investigate the effects of prostaglandin F-2α (PGF-2α) on the expression of vascular endothelial growth factor (VEGF) in human adipose tissue derived MSCs. Materials and Methods: In this experimental research, human adipose tissue was digested using collagenase. The isolated MSCs cells were treated with PGF-2α (up to 5 μg/ml) and incubated for 96 hours. Cell proliferation, secretion of VEGF and cell migration were spontaneously assayed by MTT, BrdU, ELISA, RT-PCR and scratching methods. Results: Cell growth at 1.0, 2.5, 5 μg/ml of PGF-2α was not significantly reduced compared to control cells, suggesting that these concentrations of PGF-2α are not toxic to cell growth. The results of the BrdU incorporation assay indicated that, in comparison to untreated cells, BrdU incorporation was respectively 1.08, 1.96, 2.0 and 1.8 fold among cells treated with 0.1, 1.0, 2.5 and 5.0 μg/ml of PGF-2α. The scratching test also demonstrated a positive influence on cell proliferation and migration. Cells treated with 1.0 μg/ml of PGF-2α for 12 hours showed the highest relative migration and coverage in comparison to untreated cells. Quantitative VEGF ELISA and RTPCR results indicated an increase in VEGF expression and secretion in the presence of PGF-2α. The amount of VEGF produced in response to 0.1, 1.0, 2.5 and 5.0 μg/ml of PGF-2α was 62.4 ± 3.2 , 66.3 ± 3.7, 53.1 ± 2.6 and 49.0 ± 2.3 pg/ml, respectively, compared to the 35.2 ± 2.1 pg/ml produced by untreated cells. Conclusion: Stimulation of VEGF secretion by PGF-2α treated MSCs could be useful for the induction of angiogenesis in tissue engineering in vitro.

278-283

Authors: Mohammad Amin Behmanesh, Hosein Najafzadehvarzi, Seyyedeh Mahsa Poormoosavi

Objective: Bisphenol A (BPA), an endocrine-disrupting chemical, has been considered as a possible risk factor for fertility because it induces testicular toxicity. Thus, we sought to analyze the effect of Aloe vera as plant with antioxidant properties on tissues and oxidative stress parameters in male rats. Materials and Methods: In this experimental study, 50 adult male Wistar rats (200 ± 20 g) have been used in this 56 day study. Animals were completely randomized and divided into five groups: A1 (control), A2 (vehicle control), A3 (Aloe vera gel 300 mg/kg), B1 (BPA 20 μg/kg bw) and B2 (Aloe vera gel+ BPA). At the end of the study, the rats were anesthetized and 2 ml blood samples were obtained for evaluation of oxidative stress markers. Also, both testes were collected for histological examinations. Results: BPA significantly decreased (P<0.05) body and testis weights. Seminiferous tubule diameter (STD) and height of seminiferous epithelium (HSE), were significantly decreased (P<0.05) in the groups receiving BPA as compared to the control. There was also a reduction in the quantity of spermatocyte and spermatids. Moreover, malondialdehyde (MDA) increased and thiol protein (G-SH) decreased. But, co-administration of Aloe vera with BPA accelerated the total antioxidant capacity and testicular tissue structure healing. Conclusion: According to our findings, Aloe vera gel extract can overcome the damaging effects of BPA on the reproductive system of rats and protects rats’ testes against BPA-induced toxicity.

290-292

Authors: Emad Nikkhah, Reza safaralizadeh, Javad mohammadiasl, Maryam Tahmasebi birgani, Mohammad Ali Hosseinpour Feizi, Neda Golchin

Norrie disease (ND) is a rare X-linked recessive disorder, which is characterized by congenital blindness and, in several cases, accompanied with mental retardation and deafness. ND is caused by mutations in NDP, located on the proximal short arm of the X chromosome (Xp11.3). The disease has been observed in many ethnic groups worldwide, however, no such case has been reported from Iran. In this study, we present the molecular analysis of two patients with ND and the subsequent prenatal diagnosis (PND). Screening of NDP identified a hemizygous missense mutation (p.Ser133Cys) in the affected male siblings of the family. The mother was the carrier for the mutation (p.Ser133Cys). In a subsequent chorionic amniotic pregnancy, we carried out PND by sequencing NDP in the chorionic villi sample at 11 weeks of gestation. The fetus was carrying the mutation and thus unaffected. This is the first mutation report and PND of an Iranian family with ND, and highlights the importance of prenatal diagnostic screening of this congenital disorder and relevant genetic counseling.

10-18

Authors: Nasrin Khanmohammadi, Hamid Reza Sameni,Moslem Mohammadi, Abbas Pakdel,Majid Mirmohammadkhani, Houman Parsaie, Sam Zarbakhsh

Objective: Although stem cell transplantation has beneficial effects on tissue regeneration, but there are still problems such as high cost and safety issues. Since stem cell therapy is largely dependent on paracrine activity, in this study, utilization of transplantation of bone marrow stromal cells (BMSCs)-secretome instead of the cells, into damaged ovaries was evaluated to overcome the limitations of stem cell transplantation. Materials and Methods: In this experimental study, BMSCs were cultured and 25-fold concentrated conditioned medium (CM) from BMSCs was prepared. Female rats were injected intraperitoneally with cyclophosphamide (CTX) for 14 days. Then, BMSCs and CM were individually transplanted into bilateral ovaries, and the ovaries were excised after four weeks of treatment. The follicle count was performed using hematoxylin and eosin (H&E) staining and the apoptotic cells were counted using TUNEL assay. Ovarian function was evaluated by monitoring the ability of ovulation and the levels of serum estradiol (E2) and follicle-stimulating hormone (FSH). Results: Evaluation of the ovarian function and structure showed that results of secretome transplantation were almost similar to those of BMSCs transplantation and there was no significant differences between them. Conclusion: BMSCs-secretome is likely responsible for the therapeutic paracrine effect of BMSCs. Stem cell secretome is expected to overcome the limitations of stem cell transplantation and become the basis of a novel therapy for ovarian damage.

25-30

Authors: Ahad Khoshzaban, Peyman Keyhanvar, Elham Delrish,Farhood Najafi, Saeed Heidari Keshel, Ikuya Watanabe, Alireza Valanezhad, Tahereh Jafarzadeh Kashi

Objective: Alginate, known as a group of anionic polysaccharides extracted from seaweeds, has attracted the attention of researchers because of its biocompatibility and degradability properties. Alginate has shown beneficial effects on wound healing as it has similar function as extracellular matrix. Alginate microcapsules (AM) that are used in tissue engineering as well as Dulbecco’s modified Eagle’s medium (DMEM) contain nutrients required for cell viability. The purpose of this research was introducing AM in medium and nutrient reagent cells and making a comparison with control group cells that have been normally cultured in long term. Materials and Methods: In this experimental study, AM were shaped in distilled water, it was dropped at 5 mL/hours through a flat 25G5/8 sterile needle into a crosslinking bath containing 0.1 M calcium chloride to produce calcium alginate microspheres. Then, the size of microcapsules (300-350 μm) were confirmed by Scanning Electron Microscopy (SEM) images after the filtration for selection of the best size. Next, DMEM was injected into AM. Afterward, adiposederived mesenchymal stem cells (ADSCs) and Ringer’s serum were added. Then, MTT and DAPI assays were used for cell viability and nucleus staining, respectively. Also, morphology of microcapsules was determined under invert microscopy. Results: Evaluation of the cells performed for spatial media/microcapsules at the volume of 40 μl, showed ADSCs after 1-day cell culture. Also, MTT assay results showed a significant difference in the viability of sustained-release media injected to microcapsules (P<0.05). DAPI staining revealed living cells on the microcapsules after 1 to 7-day cell culture. Conclusion: According to the results, AM had a positive effect on cell viability in scaffolds and tissue engineering and provide nutrients needed in cell therapy.

41-45

Authors: Esmaeil Rahimi, Amirhossein Ahmadi, Mohammad Ali Boroumand, Bahram Mohammad Soltani, Mehrdad Behmanesh

Objective: ANRIL is an important antisense noncoding RNA gene in the INK4 locus (9p21.3), a hot spot region associated with multiple disorders including coronary artery disease (CAD), type 2 diabetes mellitus (T2DM) and many different types of cancer. It has been shown that its expression is dysregulated in a variety of immune-mediated diseases. CAD is a major problem in T2DM patients and the cause of almost 60% of deaths in these patients worldwide. The aim of the present study was to compare the expression level of ANRIL between T2DM patients with and without CAD. Materials and Methods: In this case-control study, we examined ANRIL expression in peripheral blood mononuclear cell samples by quantitative reverse transcription- polymerase chain reaction (RT-qPCR) in 64 T2DM patients with and without CAD (33 CAD+ and 31 CAD- patients respectively, established by coronary angiography). Results: Expression analysis revealed that ANRIL was up regulated (2.34-Fold, P=0.012) in CAD+ versus CADdiabetic patients. Data from receiver operating characteristic (ROC) curve analysis has shown that ANRIL could act as a potential biomarker for detecting CAD in diabetic patients. Conclusion: The expression level of ANRIL is associated with presence of CAD in diabetic patients and could be considered as a potential peripheral biomarker.

53-60

Authors: Tae Woo Kim, Myung Chul Lee, Hyun Cheol Bae, Hyuk-Soo Han

Objective: Coculture of chondrocytes and mesenchymal stem cells (MSCs) has been developed as a strategy to overcome the dedifferentiation of chondrocytes during in vitro expansion in autologous chondrocyte transplantation. Synovium-derived stem cells (SDSCs) can be a promising cell source for coculture due to their superior chondrogenic potential compared to other MSCs and easy accessibility without donor site morbidity. However, studies on coculture of chondrocytes and SDSCs are very limited. The aim of this study was to investigate whether direct coculture of human chondrocytes and SDSCs could enhance chondrogenesis compared to monoculture of each cell. Materials and Methods: In this experimental study, passage 2 chondrocytes and SDSCs were directly cocultured using different ratios of chondrocytes to SDSCs (3:1, 1:1, or 1:3). glycosaminoglycan (GAG) synthetic activity was assessed using GAG assays and Safranin-O staining. Expression of chondrogenesis-related genes (collagen types I, II, X, Aggrecan, and Sox-9) were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry staining. Results: GAG/DNA ratios in 1:1 and 1:3 coculture groups were significantly increased compared to those in the chondrocyte and SDSC monoculture groups. Type II collagen and SOX-9 were significantly upregulated in the 1:1 coculture group compared to those in the chondrocyte and SDSC monoculture groups. On the other hand, osteogenic marker (type I collagen) and hypertrophic marker (type X collagen) were significantly downregulated in the coculture groups compared to those in the SDSC monoculture group. Conclusion: Direct coculture of human chondrocytes and SDSCs significantly enhanced chondrogenic potential, especially at a ratio of 1:1, compared to chondrocyte or SDSC monocultures.

73-77

Authors: Fahimeh Mollaahmadi, Ashraf Moini, Reza Salman Yazdi, Mehrdad Behmanesh

Objective: Infertility is a common human disorder which is defined as the failure to conceive for a period of 12 months without contraception. Many studies have shown that the outcome of fertility could be affected by DNA damage. We attempted to examine the association of two SNPs (rs1127354 and rs7270101) in ITPA, a gene encoding a key factor in the repair system, with susceptibility to infertility. Materials and Methods: This was a case-control study of individuals with established infertility. Blood samples were obtained from 164 infertile patients and 180 ethnically matched fertile controls. Total genomic DNA were extracted from whole blood using the standard salting out method, and stored at -20˚C. Genotyping were based on mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in which PCR products were digested with the XmnI restriction enzyme and run on a 12% polyacrylamide gel. Results: All genotype frequencies in the control group were in Hardy-Weinberg equilibrium. A significant association (in allelic, recessive and dominant genotypic models) was observed between infertile patients and healthy controls based on rs1127354 (P=0.0001), however, no significant association was detected for rs7270101. Also, gender stratification and analysis of different genotype models did not lead to a significant association for this singlenucleotide polymorphis (SNP). Conclusion: ITPA is likely to be a genetic determinant for decreased fertility. To the best of our knowledge, this is the first report demonstrating this association, however, given the small sample size and other limitations, genotyping of this SNP is recommended to be carried out in different populations with more samples.

84-89

Authors: Oranous Bashti, Mehrdad Noruzinia, Masoud Garshasbi, Morteza Abtahi

Objective: Endometriosis is a prevalent gynecologic disease affecting 10% of women in reproductive age. Endometriosis is diagnosed by laparoscopy that was followed by histologic confirmation. Early diagnosis will lead to a more effective treatment with much less morbidity. As miR-31 and miR-145 are shown to be directly or indirectly correlated to biological processes involved in endometriosis, the aim of this study was to examine the association of miR-31 and miR-145 expression in plasma with the presence of endometriosis. Materials and Methods: In this case control study, the plasma samples of 55 patients with endometriosis and 23 women without endometriosis were collected, extracted and analyzed by real time quantitative polymerase chain reaction (qPCR) for the expression of miR-145 and miR-31. Results: Our findings showed that miR-31 expression levels in stage 3 or 4 and stage 1 or 2 were significantly downregulated (less than 0.01-fold, P<0.05), while the expression level of miR-145 was significantly up-regulated in women with endometriosis in stage 1 or 2. Conclusion: Different cellular biological processes, such as differentiation, proliferation, mitochondrial function, reactive oxygen species (ROS) production, invasion and decidualization, are deregulated in endometriosis. miR-31 and miR-145 are microRNAs (miRNAs) with potential roles, as shown in pathologies like cancers. We found that miR- 31 was under-expressed in patients with endometriosis, while miR-145 was over-expressed in stage 1 or 2, indicating that they were relatively down-regulated in the more severe forms. Our findings suggested that these two miRNAs may be considered as potential biomarkers with probable implications in early diagnosis and even follow-up of patients with endometriosis.

98-107

Authors: Mohammad Hadi Sekhavati, Sayed Morteza Hosseini, Mojtaba Tahmoorespur, Kamran Ghaedi, Farnoosh Jafarpour, Mehdi Hajian, Kyanoosh Dormiani, Mohammad Hossain Nasr-Esfahani

Objective: The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection. Materials and Methods: In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches. Results: PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique. Conclusion: The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.

113-119

Authors: Mahsa Rezaeepoor, Mazdak Ganjalikhani-hakemi, Shima Shapoori, Nahid Eskandari, Mohammadreza Sharifi, Masoud Etemadifar, Marjan Mansuorian

Objective: Semaphorin-3A (SEMA3A) and its receptors are found on some immune cells and act as suppressors of immune cells over-activation. Considering the role of SEMA3A and its down-regulation in some autoimmune diseases, as well as our bioinformatics predictions, we assumed that miR-145-5p might affect SEMA3A expression. So, we aimed to determine the effect of miR-145-5p on SEMA3A gene expression level. Materials and Methods: In this experimental study, we evaluated the effect of miR-145-5p transfection on SEMA3A expression in peripheral blood mononuclear cells (PBMCs) using ELISA and quantitative real-time polymerase chain reaction (PCR) methods. Results: Our results showed that miR-145-5p is able to decrease SEMA3A expression at both protein and mRNA levels. These data confirmed our previous bioinformatic prediction about the inhibitory effect of miR-145-5p on SEMA3A expression. Conclusion: These results enlightened us about an unknown aspect of SEMA3A role in some autoimmune disorders like multiple sclerosis (MS) and rheumatoid arthritis (RA) and also proposed SEMA3A as a potential therapeutic approach.

127-131

Authors: Farah Talebi, Farideh Ghanbari Mardasi, Javad Mohammadi Asl, Saeed Tizno, Marziye Najafvand Zadeh

Autosomal recessive non-syndromic hearing loss (ARNSHL) is defined as a genetically heterogeneous disorder. The aim of the present study was to screen for pathogenic variants in an Iranian pedigree with ARNSHL. Next-generation targeted sequencing of 127 deafness genes in the proband detected two novel variants, a homozygous missense variant in PTPRQ (c.2599 T>C, p.Ser867Pro and a heterozygous missense variant in MYO1A (c.2804 T>C, p.Ile935Thr), both of which were absent in unaffected sibs and two hundred unaffected controls. Our results suggest that the homozygous PTPRQ variant maybe the pathogenic variant for ARNSHL due to the recessive nature of the disorder. Nevertheless, the heterozygous MYO1A may also be involved in this disorder due to the multigenic pattern of ARNSHL. Our data extend the mutation spectrum of PTPRQ and MYO1A, and have important implications for genetic counseling in unaffected sibs of this family. In addition, PTPRQ and MYO1A pathogenic variants have not to date been reported in the Iranian population.

528-536

Authors: Bahareh AMin, Reyhaneh Noorani, Bibi Marjan Razavi, Hossein Hosseinzadehh

Objective We examined the protective effects of ethanolic extract of Lippia citriodora (L. citriodora) on rats subjected to chronic constriction injury (CCI) of sciatic nerve and possible mechanisms of actions. Materials and Methods In this experimental study, the extract was administered 50, 100 and 200 mg/kg, Intraperitoneally (I.P) from the surgery time for 14 consecutive days. The changes in the spinal cord levels of apoptotic factors, microglia and astroglia markers during the time course of study were assessed by western blotting on days 3, 7 and 14 post-CCI. Results CCI rats developed neuropathy evident from a marked mechanical allodynia, cold allodynia and thermal hyperalgesia on days 3, 5, 7, 10 and 14 post-CCI. A significant increase in the levels of Iba (a marker of microglia activation) and Bax (a proapoptotic factor) was observed three days after nerve injury. The levels of Iba remained high on day 7. In contrast, there was no difference in glial fibrillary acidic protein (GFAP) contents between sham and CCI animals. Treatment with the extract significantly attenuated behavioral changes associated with neuropathy. Bax/Bcl-2 and Iba1 were decreased in CCI animals treated with the extract. Conclusion The results support the evidence that microglial activation and apoptosis are correlated with pain behaviors. It is suggested that anti-allodynic and anti-hyperalgesic effects, elicited by L. citriodora, might have some degrees of association with the inhibition of microglia activation and apoptotic pathways.

545-551

Authors: Zahra Valizadeh-Arshad, Ebrahim Shahbazi, Shiva Hashemizadeh, Azadeh Moradmand, Meysam Jangkhah, Sahar Kiani

Objective Motor neuron differentiation from human embryonic stem cells (hESCs) is a goal of regenerative medicine to provide cell therapy as treatments for diseases that damage motor neurons. Most protocols lack adequate efficiency in generating functional motor neurons. However, small molecules present a new approach to overcome this challenge. The aim of this research is to replace morphogen factors with a cocktail of efficient, affordable small molecules for effective, low cost motor neuron differentiation. Materials and Methods In this experimental study, hESCs were differentiated into motor neuron by the application of a small molecule cocktail that consisted of dorsomorphin, A8301, and XAV939. During the differentiation protocol, we selected five stages and assessed expressions of neural markers by real-time polymerase chain reaction (PCR), immunofluorescence staining, and flow cytometry. Motor neuron ion currents were determined by whole cell patch clamp recording. Results Immunofluorescence staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) indicated that they were positive for NESTIN (92.68%), PAX6 (64.40%), and SOX1 (82.11%) in a chemically defined adherent culture. The replated (hESC)-derived NE differentiated cells were positive for TUJ1, MAP2, HB9 and ISL1. We evaluated the gene expression levels with real-time reverse transcriptase-PCR at different stages of the differentiation protocol. Voltage gated channel currents of differentiated cells were examined by the whole-cell patch clamp technique. The hESC-derived motor neurons showed voltage gated delay rectifier K+, Na+ and Ca2+ inward currents. Conclusion Our results indicated that hESC-derived neurons expressed the specific motor neuron markers specially HB9 and ISL1 but voltage clamp recording showed small ionic currents therefore it seems that voltage gated channel population were inadequate for firing action potentials.

559-568

Authors: Faezeh Maghsood, Abbas Mirshafiey, Mohadese M. Farahani, Mohammad Hossein Modarressi, Parvaneh Jafari, Elahe Motevaseli

Objective Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) from Lactobacillus acidophilus (L. acidophilus) and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Materials and Methods In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RT- PCR). The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. Results Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 (P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged. Conclusion Our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response.

578-584

Authors: Ali Karimi, Rasoul Shahrooz, Rahim Hobbenaghi, Rahim Mohammadi, Esmaeil Mortaz

Objective Therapeutic angiogenesis is employed to induce vascular network formation and improve functional recovery in ischemia. The aim of this study is to find an appropriate method to recover local ischemic conditions. Materials and Methods In this experimental survey, 20 male Wistar rats weighing approximately 200-250 g were randomly divided into four experimental groups respectively: ischemia group in which the femoral artery was transected; phosphate buffer solution group (PBS) in which the femoral artery transected location was immersed with PBS; chitosan (CHIT) group in which the transected location was immersed in a 50 µL CHIT solution; and mast cell transplanted group in which the transected location was immersed with a mixture of 50 µL CHIT and 50 µL PBS that contained 1×106 mast cells. Results On day 14 after surgery, mean numbers of blood vessels of different sizes in the CHIT/mast cell group significantly increased compared to the other experimental groups (P<0.05). Conclusion Our data suggest that mast cell reconstitution could offer a new approach for therapeutic angiogenesis in cases of peripheral arterial diseases.

599-606

Authors: Fatemeh Moghani-Ghoroghi, Ghazaleh Moshkdanian, Mojtaba Sehat, Seyed Noureddin Nematollahi-Mahani, Iraj Ragerdi-Kashani, Parichehr Pasbakhsh

Objective Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. Materials and Methods In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant. Results Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. Conclusion Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudo- pregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice.

614-619

Authors: Nasrin Gharenaz Majidi, Mansoureh Movahedin, Zohreh Mazaheri

Objective Re-vitrification of embryos immediately after thawing or after a culture period could be used to preserve the extra embryos after embryo transfer. This study aims to clarify the effect of re-vitrification on Bax and Bcl-2 gene expressions of compact and early blastocyst stage embryos. Materials and Methods This experimental study was performed on mouse embryos. We collected 8-cell stage embryos (n=400) from female mature mice, 60-62 hoursafter injection of human chorionic gonadotropin (hCG). The embryos were divided into 5 groups: fresh (n=80), vitrified at the 8-cell stage (n=80), vitrified at the blastocyst stage (n=80), vitrified at the 8-cell stage, and re-vitrified at the compact (n=80) and early blastocyst stages (n=80). Embryos were vitrified by cryolock. We analyzed the developmental rates of the vitrified-warmed embryos with the chi-square test. Quantitative polymerase chain reaction (qPCR) data were analyzed with SPSS version 16 using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P<0.05 were considered statistically significant. Results The expanded blastocyst formation rate showed a significant difference in re-vitrified embryos compared with fresh embryos (P<0.05). However, this result was similar between the two re-vitrified groups. Our data showed a significant difference in expression of the Bax and Bcl-2 genes between re-vitrified and fresh embryos (P<0.05). Expressions of the Bax and Bcl-2 genes showed no significant difference between the two re-vitrified groups. Conclusion Based on our study, re-vitrification could affect developmental rate and expressions of the Bax and Bcl2 genes.

627-633

Authors: Neda Rezvan, Ashraf Moini, Sattar Gorgani-Firuzjaee, Mohammad Javad Hosseinzadeh-Attar

Objective Polycystic ovary syndrome (PCOS), an ovarian-pituitary axis androgen disorder, is a common endocrine disease in women. Obesity-induced androgenesis and imbalance of adipokine secretion may lead to some metabolic features of PCOS. The beneficial effects of polyphenolic compounds such as quercetin have been reported, however, the underlying molecular mechanism is not entirely understood. In the present study, we investigated the effect of quercetin supplementation on the expression of adiponectin receptors at the transcript level in peripheral blood mononuclear cells (PBMC) samples of PCOS patients. Materials and Methods In this randomized clinical trial, 84 PCOS subjects were randomly assigned to two groups; the treatment group received 1 g quercetin (two 500 mg capsules) daily for 12 weeks and the control group received placebo. To examine the effect of quercetin supplementation on PCOS patients in addition to biochemical and anthropometric assessments, the expression of ADIPOR1 and ADIPOR2 at the transcript level and AMPK level were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and ELISA assays respectively. Results Oral quercetin supplementation significantly increased ADIPOR1 and ADIPOR2 transcript expression by 1.32- and 1.46-fold respecetively (P<0.01). In addition, quercetin supplementation enhanced AMPK level by 12.3% compared with the control group (P<0.05). Conclusion Oral quercetin supplementation improves the metabolic features of PCOS patients by upregulating the expression of adiponectin receptors and AMPK (Registration Number: IRCT2013112515536N1).

640-646

Authors: Yu-Jing Liao, Yi-Shiou Chen, Ja-Xin Lee, Lih-Ren Chen, Jenn-Rong Yang

Objective The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. Materials and Methods In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Results Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. Conclusion The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

654-659

Authors: Tahereh Foroutan, Aisan Farhadi, Saeed Abroun, Bahram Mohammad Soltani

Objective Umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation between p53 (tumor suppressor gene) and miR-145 (suppressor of cell growth) upregulation. In this study, we have assessed how adipose-derived stem cells (ADSCs) affect the expansion of hematopoietic stem cells (HSCs), as well as miR-145 and p53 expressions. Materials and Methods In this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105. Ex vivo cultures of cordblood CD34+cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer (Thin Cert™ plate with a 0.4 µm pore size), and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction (PCR) was used to determine the expressions of the p53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide (PI) was performed to detect the rate of apoptosis in the expanded cells. Results ADSCs tested positive for mesenchymal stem cell (MSC) markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer. p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs. Conclusion ADSCs might increase HSCs proliferation and self-renewal through miR-145, p53, and their relationship.