Tehran University Medical Journal

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Background: Adipose-derived stem cells (ADSCs) have noticeable self-renewal ability and can differentiate into several cell lines such as adipocytes, osteoblasts, chondrocytes, and myocytes. Progesterone plays a significant role in the myelination of peripheral nerves. Regarding the role of progesterone on the myelination of peripheral nervous system, we evaluated its effects on the in-vitro expression of P0, S100 and Krox20 mRNA in adipose-derived stem cells. Methods: In this experimental study, rat adipose-derived stem cells were isolated from the inguinal region of the animals and were evaluated by flow cytometry before culture. In preinduction phase, the cells were sequentially treated with various factors such as β- mercaptoethanol and all-trans-retinoic acid, followed by different induction mixtures. The cells were divided into four groups including two control groups (receiving either fibroblast and platelet derived-growth factors, or fibroblast growth factor, platelet derived-growth factor, forskolin and heregulin) and two experimental groups (receiving either fibroblast growth factor, platelet derived-growth factor, forskolin and progesterone, or fibroblast growth factor, platelet derived-growth factor, heregulin and progesterone). Expression of Schwann cell markers, S-100, P0 and Krox20 mRNA, was determined by semi-quantitative RT-PCR. Results: ADSCs expressed CD90, CD73, and CD31 but showed lack of CD45, and VEGFR2 expression. After the induction stage, S-100, P0 and Krox20 mRNA were expressed in the progesterone receiving experimental groups, but expression of S-100 and Krox20 mRNA were less than the control group which was receiving forskolin and heregulin (P<0.0001). Conclusion: Progesterone can promote the in-vitro expression of S-100, P0, and Krox20 genes in adipose-derived stem cells

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Background: Studies indicate that obestatin, an anti-hunger peptide, plays an important role in energy balance, GH secretion, and body weight. It has been physiologically shown that obestatin apposes the function of Ghrelin. The purpose of the present study was to investigate the effects of a single session of aerobic exercise in trained women (a 1.5-mile run) on the expression of obestatin gene found in lymphocytes.

Methods: 16 trained female participants (4±1 years of training experience) were voluntarily selected from Khorasan province in Iran and were randomly divided into two groups: the control and aerobic exercise groups. The participants in the aerobic group were asked to run for 1.5 miles with a fixed speed (70 VO2 max) while the controls were passively present in the exercise environment. Following an overnight fast, blood samples (10 ml from the antecubital vein) were collected before and immediately after the exercise from all the participants. Obestatin expression was investigated after separating the lymphocytes by centrifuge and using semi-quantitative RT-PCR.

Results: There was a rise in obestatin gene expression in the case group after one session of aerobic training versus the control group but the changes were not statistically significant.

Conclusion: The results indicated that a single aerobic exercise could not significantly increase the expression of obestatin. Perhaps the type, duration and intensity of the applied protocol in this study did not have a cumulative effect on this gene although these results are in harmony with the results of other studies in this regard.

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Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 Background: The techniques used in assisted reproductive technologies have progressed considerably, but many embryos do not implant after transfer upon the use of these techniques. One of the causes of infertility is repeated implantation failure due to decreased endometrial receptivity. Furthermore, in clinical conditions such as endometriosis and myoma, implantation decreases after embryo transfer. In this case-control study the expression patterns of HOXA-10 and BTEB1 mRNAs were evaluated at the time of implantation in patients with myoma and endometriosis.
Methods : In this study performed in Hamadan University of Medical Sciences during 1389, the cases included 16 patients with endometriosis and myoma (8 in each group) and the control group consisted of 8 fertile women. Endometrial sampling was performed at mid-secretory phase. Later, the expression patterns of HOXA-10 and BTEB1 mRNAs were evaluated using a semi-quantitative RT-PCR method.
Results : The optimal PCR cycles determined were 30, 32 and 26 for HOXA10, BTEB1 and β-actin, respectively. Endometrial HOXA-10 and BTEB1 mRNA expression levels (normalized to ß-actin expression) at the time of implantation were significantly decreased in the endometrium of infertile patients with endometriosis compared with that of healthy fertile controls (P<0.05). A similar pattern was seen in patients with myomas for both HOXA10 and BTEB1 genes, (P<0.05).
Conclusion: It seems that lower expression of HOXA-10 and BTEB1 mRNAs in the implantation window of endometrium that increase normally, could account for some aspects of infertility in patients with endometriosis and myoma.

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Background: Several dietary factors are involved in cardiovascular coronary heart diseases, including trans fatty acids, which are generally formed during hydrogenation of vegetable oils, a process that causes conversion of liquid oils into semisolid fats. Nowadays, it is well-known that trans fatty acids form a major risk factor in the occurrence and progression of atherosclerosis. On the other hand, it has been identified that some nuclear receptors, such as PPARs,are involved and play important roles in lipid homeostasis and pathogenesis of cardiovascular diseases. Therefore, we studied the effect of elaidic acid on gene expression of peroxisome proliferator activated receptor gamma (PPARγ).

Methods: Murine macrophage RAW264.7 cells were treated by 0.5, 1, and 2 mM concentrations of elaidic acid for 6 h. The control group was treated by 50% ethanol (as solvent), equivalent to the amount of ethanol used in 2 mM concentration of elaidic acid. Later, the total RNA was extracted and its cDNA was synthesized. Finally, the quantity of PPARγ gene expression was measured by real-time PCR.
Results:  Overall,0.5, 1, and 2 mM concentrations of elaidic acid decreased PPARγ gene expression in RAW264.7 macrophage cell line by -1.36, -1.68, and -3.24 folds compared with the control group, respectively.
Conclusion: By decreasing the expression of nuclear receptor PPARγ, elaidic acid causes, intensifies or accelerates the occurrence of cardiovascular diseases, especially atherosclerosis.This finding shows the importance of reducing the consumption of elaidic acid containing foods.

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Background: Colorectal cancer is the third most common cancer in the world. Non-coding RNA especially miRNAs have important regulatory roles in cancer. miRNAs are small non coding RNA 21-23 nucleotides long which have different levels of expression between tumors and normal tissues. This study was designed to compare expression level of miRNA-21 between Iranian population colorectal cancer tissues and normal tissue. Methods: This case-control study has performed in medical genetics department of Tehran University of Medical Sciences from January to November 2013. We used 35 samples. The samples were isolated from tumor and adjacent normal tissues of colon. Thirty-five samples were divided into different groups according to cliniopathologic features including tumor size (>4 and <4 cm), metastasis (+ and -) and stage. After small RNA extraction from tissues by small RNA purification kit the quality and quan-tity of extracted RNA was determined using spectrophotometry. cDNAs were synthe-sized and real-time polymerase chain reaction carried out. Finally expression levels were statistically analyzed by LinRegPCR and REST software. Results: miRNA-21 expression ratio in stages I, II and III were 1/804 and 4/574, re-spectively, the increase from stage III was statistically significant (P= 0.037). The ex-pression were also studied according to different clinicopathologic status of colon can-cer, tumor size (>4 and <4 cm) and metastatic (+ and -), miRNA-21 over expressed in both groups, however the increase was not statistically significant. Conclusion: In this study, we found miR-21 over-expression in advanced stage in tu-moral tissue comparing with normal adjacent tissue. This means perhaps in the future it would be possible to use miRNA-21 as an informative prognostic biomarker to guide for better treatment strategies for colorectal cancer patients. Our findings also indicate that miRNA-21 is a promising new molecular target for designing novel therapeutic strategies to control colorectal cancer.

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Background: About 15% of couples have fertility problems and male factor in fertility accounts for half of the cases. In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESCs) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Testis specific 10 (Tsga10) during this process. Methods: This is an in vitro study that was performed in department of Medical Genetics in Tehran University of Medical Sciences from February 2012 to March 2013. Mouse embryonic stem cells were cultured on mouse embryonic fibroblast as feeder layer. Then mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes (Stra8 as pre-meiotic, Dazl and Sycp3‌ as meiotic and Protamin1 and Spata19 as Post-meiotic). Expression of Testis Specific Gene 10 (Tsga10) at RNA and protein levels was then analyzed. Results: It was shown that transition of embryonic stem cells from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until 25th day of the culture. Results showed low level of Tsga10 expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed form of the related protein was similar to those in vivo as well. Conclusion: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.

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Background: Breast cancer is the second most common cancer in the world after lung cancer also is the fifth cause of cancer mortality. About 90 percent of cancer mortality is because of metastasis and devastating between cell attachments, especially tight cell junctions. Epithelial mesenchymal transition is a phenomena involved in metastasis and starts with cell detachment. Occludin is the integral membrane protein which is located in tight junctions. Obviously distressing tight junction, which facilitates the stages of metastasis in cancer cells are very critical step. The aim of this study was to demonstrate the importance of occludin expression and its relationship with invasiveness in human breast cancer. Methods: In a cross sectional study we evaluated 30 patients who were referred to Caner Institute of Imam Khomeini Hospital Complex, Tehran, Iran, from March 2013 to April 2013. Samples were derived from fresh frozen tumor of patients suffering from breast cancer after inform consent assignment in the Tumor Bank of Iran (TBI). RNA was extracted from tumor tissue followed by reverse transcription, polymerase chain reaction (PCR), conventional Real-time PCR and data analysis for the occludin gene expression. Data were analyzed based on clinical staging of breast cancer patients which were cited in data bank of TBI. Results: Results of this study have demonstrated that the occludin gene expression was increased with the advanced stage. In 22 of patients the expression of gene was elevated compared with normal samples. On the other hand, the expression was significantly increased in stage II in comparison with stage I. Conclusion: The expression of occludin has increased by elevation of stage compared with normal tissue. It is suggested that alteration in the expression of this gene might be a possible factor which could affect on patient’s prognosis the same as other factors which are belonging to the same family. Increasing in expression of this gene might be considered as one of the possible markers which predict the possibility of invasion and metastasis.

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Cancer/Testis antigens (CTAs) as a group of tumor antigens are the novel subjects for developing cancer vaccine and immunotherapy approaches. They aberrantly express in tumors with highest normal expression in testis, and limited or no expression in normal tissues. There are important similarities between the processes of germ-cell and cancer cell development Spermatogenesis begins at puberty when expression of novel cell-surface antigens occurs when the immune system has been refined the ability to distinguish self from non-self. Whereas macrophage and lymphocytes are commonly found within interstitial spaces of the testis, these antigen-presenting cells are rarely seen within the seminiferous tubules. These observations have led to the concept of the immune privileged site for testis. Localized normal expression of the CT genes in testis that makes them immunogenic for immune system, in one side, and their abnormal expression in different kinds of cancer cells, in the other side, has make them as promising target for developing cancer vaccines and new cancer therapeutics approaches. In malignancies, gene regulation is disrupted which results aberrant expression of CT antigen in a proportion of tumors of various types. For some CTAs, data support their fundamental role in tumorigenesis. Several authors believe it is not clear whether they have an essential role in tumorigenesis or they are by-products of chromatin variations in cancer. There is a growing list of CTAs within them advanced clinical trials are running by using some of them in cancers like lung cancer, malignant melanoma and neuroblastoma. In this review we discuss the gene TSGA10 as an example of CT genes. TSGA10 expresses in its highest levels in elongating spermatids and localized in the fibrous sheath of mature sperm. This gene is proposed as a serological biomarker in cutaneous lymphoma. Its abnormal expression has been reported in different cancers such as acute lymphoblastic leukemia, breast, brain, gastrointestinal and a range of other cancers either in mRNA or protein levels. It has an important role in angiogenesis in cancer tumors because of its effects in the gene hypoxia-inducible factor (HIF1). Absence or lack of TSGA10 expression has been reported in ascosporic infertile men.

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Background: Breast cancer is the most common non- skin cancer among women and it’s the second leading cause of cancer related death in women. Ubiquitin and ubiquitin like proteins are member of signal transduction pathways which have several cellular functions. It has shown that Ubiquitin like protein D (UBD) has accelerated the cancer progress. The aims of this study is evaluation of UBD gene expression in women suffering from breast cancer and its correlation with disease progression. Methods: In this study 30 FFPE (Formalin-fixed, paraffin-embedded) samples 20 cases from breast cancer and 10 cases from mammoplasty were collected from Parsian and Kasra Hospitals in Tehran after confirmation by pathologist. For each sample collection characters included ER-positive, lymph node negative, tumor size less than 5 cm in diameter were considered. Samples belonged to May 2010 up to April 2012. At first paraffin was removed by adding xylene then xylene removed with replacing ethanol 98%. After removing ethanol, RNA was extracted from samples by using RNX plus solution and cDNA synthesis were performed by using Moloney murine leukemia virus (M-MuLV) enzyme. UBD gene expression were examined in all samples cDNA by relative Real Time PCR. In this study GAPDH gene expression was also used as internal control. Results: UBD gene expression was obtained by calculating ΔΔCT and RQ. The average incensement of UBD gene expression in comparison of normal samples was 11 times. The results have shown that the level of UBD expression was related to the development and extend of the disease. In patients with stage 1 of disease, UBD gene expression had 2.73 times increase (P=0.001) compared to the control samples. However in stage 4 of disease, this number has increased up to 19.4 times (P=0.0005) more than normal. Conclusion: Considering the results of this study, it could be said that UBD gene expression as useful biomarker has an important role in detection of breast cancer. In addition as UBD gene expression levels increased stages of disease increased too. So that evaluation of UBD gene expression can be useful in early detection of disease.

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Background: Spermatogenesis is a complex and highly organized process of proliferation and differentiation of spermatogonial stem cells. Spermatogonial stem cells (SSCs) as a unique stem cell have the potential to self-renewal, differentiation and transmit genetic information to the next generation and play a vital role in maintaining fertility. Sertoli cells as the only somatic cells within the seminiferous epithelium play central roles in the formation of niche and balance between self-renewal and differentiation by secrete many growth factors. Given the importance and widespread use of SSCs, particularly in the treatment of infertility, the aim of this study was to create an optimal environment for the proliferation of SSCs. So we decided to study of undifferentiated (ID4) and differentiated (c-Kit) gene expression in SSCs followed by co-culture with Sertoli cells for a one-month.

Methods: This experimental study was conducted from November 2013 to December 2014 in Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, on immature NMRI mouse (6-3 days old). Initially, Sertoli cells and SSCs were isolated from neonates mouse testes during the two-step enzymatic digestion characteristics Sertoli cells with vimentin marker and SSCs with promyelocytic leukemia zinc-finger (PLZF) marker were confirmed. Then SSCs were cultured in two groups: co-culture with Sertoli and without co-culture (control). Undifferentiated (ID4) and differentiation (c-Kit) gene expression were evaluated by Real-time PCR technique.

Results: Spermatogonial stem cells purity was obtained 66.91% by flow cytometry. The relative expression levels of gene ID4 in co-culture group at the end of each week, compared to the control group showed a significant increase (P<0.05). While the expression of this gene significantly decreased in each group over time (P<0.05). The results of the comparison of the relative expression of c-Kit gene in co-culture group are indicated significant decrease than the control group at the end of each week (P<0.05). In addition, this gene expression was showed significant increase in each group individually over time (P<0.05) ID4 gene expression showed a significant (P<0.05) increase toward the control group, while in the expression of c-Kit was observed a significant (P<0.05) decrease compared with the control group at the end of each week.

Conclusion: According to the results of this study, co-culture with Sertoli cells maintains SSCs in the prolifration stage for long-term, so can be used to optimize the culture medium at the clinic.

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Background: Infeliximab is a form of chimeric antibody which neutralizes the most important inflammatory cytokine, TNF-a, in inflammatory disorders. The aim of current study was to pilot expression of chimeric infliximab in Chinese Hamster ovary (CHO) cells.

Methods: In this research study, pVITRO2-neo-mcs vector that consist of infliximab light chain and heavy chain was used to transform into the E.coli by CaCl2 method. The plasmid was then purified and transfected to cultured CHO cells by Lipofectamine 2000® (Invitrogen GmbH, Germany). Transfected cells were selected upon G-418 treatment after 2 weeks and the level of expression, based on standard curve, was measured using IgG ELISA kit after 48 hours for each clone. High level expressed clone was then cultured in roller bottles and recombinant chimeric product was purified by protein A affinity chromatography. The purity of the product was analyzed by 10% gel SDS-PAGE from eluted samples. The efficacy of the purification was analyzed by ELISA before and after purification step. This article is a master's student thesis from February 2015 to August 2016 in pharmaceutical technology development center, Tehran University of Medical Sciences, Tehran, Iran.

Results: The purified plasmid was analyzed on 2% agarose gel. After selective pressure of G-418, 10 stable transfect clones were assessed for infliximab secretion by IgG ELISA kit at 450 nm. The maximum and minimum expression which detected by ELISA were 23 ng/ml and 6 ng/ml, respectively. The band width of infliximab fraction during purification procedure was observed at 0.7-0.8 min. The efficiency of the purification by ELISA was 70%. On SDS-PAGE analysis, two bands, 25 and 50 kDa, respect to light and heavy chains of Infliximab, was confirmed the expression of recombinant protein.

Conclusion: In the current study, the construct for infliximab monoclonal antibody production was designed using genetic engineering techniques and the expression was confirmed by conventional molecular biology methods. The high yield production was carried out in semi-industrial scale using roller bottles with a 70 percentage of purification efficiency.

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Background: Studies show that aerobic exercise prevents osteoporosis in menopause by stimulating osteoblastic cells. Therefore, the purpose of this study was to investigate the effect of 12 weeks of moderate-intensity aerobic exercise on alkaline phosphatase gene expression, serum levels of alkaline phosphatase, parathyroid hormone, and calcium in sedentary women.

Methods: This investigation is a semi-experimental study that was performed in September 2015 at Urmia University, Iran. The statistical population was all healthy and sedentary postmenopausal women 50 to 65 years old in Urmia city. Twenty sedentary postmenopausal women with an average age 60.12±2.12 yr, weight 72.35±10.50 kg, and body mass index 29.46±3.24 kg/m2 voluntarily and bona fide participated in this study, and then subjects were randomly divided to the Exercise/E (10 women) and Control/C (10 women) groups by random sampling method. E group performed of 12 weeks walking and jogging moderate-intensity aerobic exercise at 65-70% maximal heart rate of training, three sessions per week and per session 50-60 (min), but the C group participated in no intervention. Twenty-four hours before and after the 12-week training program were taken blood samples in order to measure of alkaline phosphatase gene expression and serum markers of bone in the E and C Groups. Evaluation of gene expression and serum markers of bone were measured by real-time reverse transcription PCR (RT-PCR) and Auto-analyzer (Biotechnica, Italy)/ ELISA reader (Awareness Inc., USA) machines, respectively. Data analysis included descriptive and inferential (ANCOVA test) statistics using SPSS version 23 (Chicago, IL, USA) and a significance level of P≥0.05 was considered. Results: The results showed that alkaline phosphatase gene expression and parathyroid hormone after 12 weeks of moderate-intensity aerobic exercise in between-groups were significantly increased (P=0.027 and P=0.006, respectively), while serum levels of calcium and alkaline phosphatase were not significantly different (P=0.941 and P=0.990, respectively).

Conclusion: The results suggest that 12 weeks of aerobic exercise of walking and jogging at 65-70% maximal heart rate of training increases alkaline phosphatase gene expression and parathyroid hormone in sedentary postmenopausal women.

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Background: As far as the role and amount of Transferrin receptor 2 (TFR2), which is the transferrin receptor gene, studies have been conducted, some of which confirming its relationship with gastric adenocarcinoma. The idea behind this study was to examine changes in the TFR2 gene expression in the tumor cells of gastric adenocarcinoma and comparing with gene expression in the normal tissue adjoining the tumor.

Methods: This case-control study was conducted at the Pathology Section of Cancer Institute of Imam Khomeini University Hospital in Tehran from September 2015 to September 2016. In this study, 30 fresh samples from tumor tissues of patients diagnosed with gastric adenocarcinoma, 30 fresh samples of normal tissue adjoining the tumor and 30 samples of frozen plasma from the same patients were taken. The patients' plasma was examined in terms of existence of helicobacter pylori antibody by enzyme linked immunosorbent assays (ELISA) method and TFR2 gene expression in the tumor tissue and the adjoining normal tissue by applying real-time polymerase chain reaction (Real-Time PCR).

Results: Gene expression (by applying real time polymerase chain reaction) in the tumor tissue was meaningfully higher than in the normal tissue (P= 0.125). The TFR2 expression in patients with stomach cancer, who were at the same time infected with helicobacter pylori, indicated that the gene expression had increased in those with this contamination (P= 0.077). Examining the relationship between this gene expression and the stage of disease showed that the TFR2 gene expression increased significantly in the more advanced stages of the disease (P= 0.396).

Conclusion: The TFR2 gene expression increases in the stomach's tumor tissue. This gene expression is higher in people infected with helicobacter pylori or in those at an advanced stage of the disease. These findings may confirm the direct relationship between gene expression and the occurrence or metastasis of gastric adenocarcinoma.

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