Cell Journal (Yakhteh)

1-6

Authors: Yousef-Reza Yousefnia Pasha , Seyed Gholam-Ali Jorsaraei , Mahtab Zeinalzadeh , Masoumeh Golsorkhtabar Amiri , Arsalan-Ali Ramaji

Objective: In sever oligospermia; one of the paths used for surgical sperm retrieval (SSR) is to extract sperm via a testicular biopsy. The aim of our study is to determine the reliable time interval between testicular biopsy and intracytoplasmic sperm injection (ICSI) procedure in order to obtain optimumsperm parameters (Count, motility and normal morphology). Materials and Methods: This cohort study was carried out on 30 patients which were candidates for ICSI. After collection and keeping the samples obtained from the testicular biopsy in Ham’s F10 environment, the concentration, motility and morphology of the sperm in each sample was evaluated immediately as well as 2 and 4 hours after processing. The Data were then compared with each other. For the statistical analysis, Friedman, Willcoxon and Cochran tests were used. Results: The mean of sperm concentration was 5.69 ± 6.14 million and the motility was10.83 ± 12.63% at 2 hours following biopsy which was significantly higher than those obtained after 0 and 4 hours of the biopsy (p <0.05). Conclusion: The reliable preincubation time which resulted in the highest rate of spermatozoa parameters after testicular biopsy and before incubation was 2 hours.

19-24

Authors: Mehri Hajikhan Mirzaei , Mehrdad Noruzinia , Hamid Karbassian , Yousef Shafeghati , Mousa Keyhanee , Ali Bidmeshki-Pour

Objective: Breast cancer is one of the most common malignancies in women worldwide. It is caused by a number of genetic and epigenetic factors. Aberrant hypermethylation of the promoter regions in specific genes is a key event in the formation and progression of breast cancers as well as the DBC2 gene, as a tumor suppressor gene. Different studies show that the DBC2 gene is inactivated through epigenetic mechanisms such as methylation in its promoter region. In this study, authors have tried to analyze methylation status in the promoter region of DBC2 gene in affected women and healthy controls. Materials and Methods: In this experimental study, we evaluated the methylation status of DBC2 gene with nested methylation-specific PCR (MSPCR) using specific methylated and unmethylated primer sets, in three separate PCR reactions. We used 50 tissue and blood samples of patients with breast cancer, 5 normal tissues and also 30 normal blood samples. Results were evaluated by the Mann-Whitney test, SPSS 16.0 statistical software. Results: Nested MSPCR results demonstrated that the frequency of the DBC2 promoter region methylation status in tumor and blood samples of the affected patients was significantly higher than that of the corresponding normal controls. Conclusion: DBC2 gene inactivation by methylation of CpG islands in the promoter region probably is a crucial step in the process of cell proliferation and susceptibility to different cancers, including breast cancer. Our study provides new evidence that aberrant methylation of the DBC2 gene is involved in the tumorigenesis of breast cancer. DNA methylation in this gene is proven to be a potential marker for tumor diagnosis and prognosis, as well as a novel therapeutic target.

31-38

Authors: Hoda Jahani , Saeid Kaviani , Majid Hassanpour-Ezatti , Masoud Soleimani , Zeinab Kaviani , Zahra Zonoubi

Objective: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared, and then p-PCL scaffolds were evaluated for MSC culture. Materials and Methods: Aligned and random PCL nanofibrus scaffolds were fabricated by electrospining and their surface modified with O2 plasma treatment to enhance MSC proliferation, adhesion and interaction. Chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance (ANOVA). Results: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber. Conclusion: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.

47-52

Authors: Atefeh Hemati , Mahnaz Azarnia , Mohammad Nabiuni , Ghadireh Mirabolghasemi , Saeed Irian

Objective: Medicinal plants are widely used throughout the world. Since these plants are known to have minimal side effects, many people embrace them. The golpar plant, scientifically known as Heracleum persicum (H. persicum), is a common Asian and Iranian medicinal plant. The use of golpar is recommended in traditional medicine as a contraceptive medication for females; however, no scientifically documented evidence has been reported. This study investigates the effects of the golpar plant on ovarian tissue and folliculogenesis. Materials and Methods: In this experimental study, H. persicum hydroalcoholic extract (HPHE) was used at 400 mg/kg and 1600 mg/kg doses. Adult female rats were divided into three groups: control, sham, and experimental(I, II). The control group did not receive any injection, the sham group received saline solution, and the experimental group received IP injections of HPHE for 21 days, once every other day, during the sexual cycle. At the end of the injection period, ovarian samples were harvested for histological studies. The FSH assay was performed according to the chemiluminescence immunoassay (CLIA) method. Data were statistically analyzed by the Instat3 program and one-way ANOVA. A p value of <0.05 was considered significant. Results: In the experimental group the numbers of primordial and primary follicles increased (p<0.001), while the number of preantral and antral follicles decreased (p<0.01). The atretic follicles decreased in the experimental group, but this decrease was not significant. There was no statistical difference in FSH concentration when compared with the control group. Conclusion: This report gives primary information on the in vivo effects of the HPHE on the ovarian follicles of the female Wistar rat. The results suggest that administration of HPHE may have inhibitory effects on folliculogenesis and cause infertility in females.

61-66

Authors: Maryam Sadat Nabavinia , Mahboobeh Naderi Nasab , Zahra Meshkat , Mohammad Derakhshan , Mehrangiz Khaje-Karamadini

Objective: Despite using the Bacille Calmette Guerin (BCG) vaccine, tuberculosis (TB) is still a worldwide disease that kills 2-3 million people each year. Developing a new and more effective vaccine is one way to possibly reduce the morbidity and mortality of TB. The Mtb72F vaccine is one of the important subunit vaccines applied in human clinical trials. In this study, we have constructed an expression vector that contains the Mtb72F fragment with some new modifications. Materials and Methods: In this experimental study, Mtb32N and Mtb39 fragments were amplified by polymerase chain reaction (PCR) using specific primers and inserted into pET21b\Mtb32C. Colony-PCR, restriction enzyme analysis, and DNA sequencing were used to confirm the accuracy of the cloning. We used Western blot to verify the desired protein expression. Results: The amplified fragments showed the desired size in PCR and digestion methods, and protein expression was confirmed using a monoclonal antibody. Conclusion: Our modification made it possible to insert another gene or gene fragments into the Mtb72F vector for developing new constructs. In addition, our data has shown that the placement of the histidine tag in the carboxyl- (C-) or amino- (N-) terminal part of a protein may influence protein expression and/or stability.

76-81

Authors: Touraj Farazmandfar , Hossein Khanahmad Shahreza , Mohammad Reza Haghshenas , Ghasem Janbabai , Hossein Azadeh , Nader Mansour Samaei

Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP) gene expression by a fluorescence microscopy. Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

98-101

Authors: Majid Farshdousti Hagh , Mehrdad Noruzinia , Yousef Mortazavi , Masoud Soleimani , Saeid Kaviani , Maryam Mahmodinia Maymand

Objective: Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells (MSCs) has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix (OSX) promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs. Materials and Methods: In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5μM Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection (MOI) treatment, gene specific methylation of OSX was analyzed by methylation specific PCR (MSP). Results: The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation (p<0.05), but expression of OSX didn’t change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards. Conclusion: MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases.

102-109

Authors: Pantea Izadi , Mehrdad Noruzinia , Morteza Karimipoor , Mohammad Hamid Karbassian , Mohammad Taghi Akbari

Objective: Breast Cancer is the most common cancer in Iranian women. Breast tumors are classified based on the estrogen receptor alpha (ERα) expression status into ER negative and ER positive tumors. ER negative tumors tend to have worse prognosis and less likely to respond to endocrine therapy. Aberrant methylation of gene promoter is one of the mechanisms for gene silencing in breast tumors. Because of its reversible nature, promoter methylation is a good target for new therapeutic strategies. We aimed to evaluate the frequency of this epigenetic event in ERα gene and its association to clinicopathological features in Iranian breast cancer patients. Materials and Methods: In this case control study the patient series consisted of 100 sporadic primary breast cancer cases (51 ER negative and 49 ER positive tumors). None of the participants had chemo or radiotherapy before surgery. In breast tumors ERα promoter methylation were assessed with methylation specific polymerase chain reaction (MSP). Data was collected on clinicopathological features of the patients. Correlation between ERα methylation and clinicopathological characteristics of the patients was investigated by Pearson Chi-Square and Fisher’s exact test. Results: ERα methylation was detected in 98% of ER negative and 65% of ER positive breast tumors. A strong correlation was found between ERα methylation and ER negativity in tumors (p<0.0001). Also, ERα methylation has associated to progesterone receptor negativity (p<0.008) and double receptor negative status (p<0.0001) in breast tumors. Conclusion: ERα methylation occurs with high frequency in the breast tumors of Iranian breast cancer patients and may play a considerable role in pathogenesis of ERα negative tumors as a poor prognosis and more aggressive category. The reversible nature of DNA methylation may provide new therapeutic possibilities in ER negative breast tumors.

116-121

Authors: Akram Hosseini , Firouz Ghaderi Pakdel , Abbas Ahmadi , Samad Zare Ph.D

Objective: This study aims to evaluate the protective effects of American ginseng administered by gastric intubation on sperm vital quality in adult male rats treated with cyclophosphamide (CP). Materials and Methods: In this experimental study, 28 Adult male Wistar rats were assigned to four groups, seven rats in each. The animals allocated to control, CP treated, Ginseng treated and CP-Ginseng treated groups. Rats were treated with CP (6.1 mg/kg/day, i.p) for 6 weeks. American ginseng was used at a dose of 500 mg/kg/day during treatment. Sperm analysis (motion, count, morphology and viability) were evaluated at the end of the experiments. Sperm motion was assessed by Computer-Assisted Sperm Analysis (CASA). The data were analyzed using GB stat software. Probability values of p<0.05 and p<0.01 were considered significant. Results: The epididymal sperm counts in the groups that received CP showed significant decreases compared to the control group. Also dead and abnormal sperms significantly increased following CP treatment compared with control. The motility of caudal sperm was reduced significantly with CP treatment. Therefore, according to the results of this study, co-administration of CP and American ginseng can improve these parameters. Conclusion: American ginseng can prevent the cytotoxic effects of CP on sperm quality factors.

130-141

Authors: Mohammad Mehdi Hassanzadeh Taheri , Ali Reza Ebrahimzadeh Bideskan , Mohammad Reza Miri

Objective: The development of vertebrae is a complex phenomenon that is correlated with distinct morphological and biochemical alterations in the paraxial mesenchyme and glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique. Materials and Methods: In this descriptive-analytic study, B4G fixed paraffin sections of 9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven different HRP-labelled lectins: Glycin max (SBA), Maclura pomifera (MPA), Wistaria floribunda (WFA), Vicia villosa (VVA) which all of them are specific for N-acetylgalactosamine (GalNAc), Ulex europius (UEA1, binds to α-L-fucose), wheat germ agglutinin (WGA, binds to sialic acid), and Griffonia simplicifolia (GSA1-B4, binds to galactose terminal sugars). The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins’ reactions with the specimen, from negative (-) to severe (+++). Data was analysed with SPSS software (version 11.5) and the non-parametric Kruskal Wallis test; p<0.05 was considered significant. Results: Our findings showed that among the tested lectins, only GalNAc residue sensitive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continued to GD15 constantly, while MPA reactions continued strongly to GD12, significantly decreased thereafter (p<0.001), and then disappeared. VVA and SBA bindings initiated weakly on GD10 and continued to GD12 without changing. These reactions increased significantly (p<0.001) thereafter, became severe to GD14, and later disappeared. The other tested lectins did not reveal regulated changes. Conclusion: According to these findings it can be concluded that only the GalNAc terminal sugar showed temporally regulated changes during the early embryonic development of vertebrae in mice. Therefore it most likely plays a key role (s) in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other tested terminal sugars may have no role in this phenomenon.

161-170

Authors: Mohammad Hussein Abnosi , Zahra Jafari Yazdi

Objective: Although arsenite is toxic it is currently recommended for the treatment of malignancies. In this study the effects of sub-micromolar concentrations of sodium arsenite on the viability, morphology and mechanism of cell death of rat bone marrow mesenchymal stem cells (BMCs) over 21 days was investigated. Materials and Methods: In this experimental study, BMCs were extracted in Dulbecco’s Modified Eagles Medium (DMEM) containing 15% of fetal bovine serum (FBS) and expanded till the 3rd passage. The cells were treated with 1, 10, 25, 50, 75 and 100 nM of sodium arsenite for 21 days and the viability of the cells estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) and trypan blue staining. Cells were then treated with the selected dose (25 nM) of sodium arsenite to determine their colony forming ability (CFA) and population doubling number (PDN). Morphology of the cells was studied using florescent dyes, and the integrity of the DNA was investigated using the comet assay and agarose gel electrophoresis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the caspase 3 assay were then applied to understand the mechanism of cell death. Data was analyzed using one way ANOVA, Tukey test. Results: A significant reduction of viability, PDN and CFA was found following treatment of BMCs with 25 nM sodium arsenite (p<0.05). Cytoplasm shrinkage and a significant decrease in the diameter of the nuclei were also seen. Comet assay and agarose gel electrophoresis revealed DNA breakage, while positive TUNEL and activated caspase 3 confirmed the apoptosis. Conclusion: A low concentration of sodium arsenite (25 nM) caused reduction of viability due to induction of apoptosis. Therefore, long term exposure to low dose of this chemical may have unwanted effects on BMCs.

177-184

Authors: Mehdi Mehdizadeh , Fataneh Dabaghian , Akram Nejhadi , Hassan Fallah-huseini , Samira Choopani , Nima Shekarriz , Nima Molavi , Arghavan Basirat , Farzaneh Mohammadzadeh Kazorgah , Alireza Samzadeh-Kermani , Sara Soleimani Asl

Objective: The spice Zingiber officinale or ginger possesses antioxidant activity and neuroprotective effects. The effects of this traditional herbal medicine on 3,4-methylenedioxymethamphetamine (MDMA) induced neurotoxicity have not yet been studied. The present study considers the effects of Zingiber officinale on MDMA-induced spatial memory impairment and apoptosis in the hippocampus of male rats. Materials and Methods: In this experimental study, 21 adult male Sprague Dawley rats (200-250 g) were classified into three groups (control, MDMA, and MDMA plus ginger). The groups were intraperitoneally administered 10 mg/kg MDMA, 10 mg/kg MDMA plus 100 mg/kg ginger extract, or 1 cc/kg normal saline as the control solution for one week (n=7 per group). Learning memory was assessed by Morris water maze (MWM) after the last administration. Finally, the brains were removed to study the cell number in the cornu ammonis (CA1) hippocampus by light microscope, Bcl-2 by immunoblotting, and Bax expression by reverse transcription polymerase chain reaction (RT-PCR). Data was analyzed using SPSS 16 software and a one-way ANOVA test. Results: Escape latency and traveled distances decreased significantly in the MDMA plus ginger group relative to the MDMA group (p<0.001). Cell number increased in the MDMA plus ginger group in comparison to the MDMA group. Down-regulation of Bcl-2 and up-regulation of Bax were observed in the MDMA plus ginger group in comparison to the MDMA group (p<0.05). Conclusion: Our findings suggest that ginger consumption may lead to an improvement of MDMA-induced neurotoxicity.

193-202

Authors: Mahboobeh Ashrafi , Seyedeh Zahra Bathaie , Saeid Abroun

Objective: N-nitroso-N-methylurea (NMU) induces breast cancer in rodents, particularly in rats. This model of breast cancer is very similar to human breast cancer. As a continuation of our recent work, we investigated the expressions of cyclin D1 and p21 in NMU-induced breast cancer of Wistar Albino rats. Materials and Methods: In this experimental study, mammary carcinoma was induced in female Wistar Albino rats by a new protocol which included the intraperitoneal injection of NMU (50 mg/kg) at 50, 65, and 80 days of the animal’s age. The animals were weighed weekly and palpated in order to record the numbers, location, and size of tumors. Subsequently tumor incidence (TI), latency period (LP), and tumor multiplicity (TM) were reported. About four weeks after the tumor size reached 1.5 cm3, rats were sacrificed. Cyclin D1 and p21 expressions in tumors and normal mammary glands from normal rats were measured by reverse-transcription polymerase chain reaction (RT- PCR) and Western blot analysis. Statistical analysis of the data was performed using SPSS software version 16.0. Results: The efficiency of tumor induction was 65%, LP was 150 days, and a TM of 1.43 ± 0.53 per rat was noted. RT-PCR and Western blot data indicated significant (p<0.05) induction of both cyclin D1 and p21 expressions in rat mammary tumors compared with normal tissue from the control group. Conclusion: These results indicate an efficient mammary tumor induction protocol for this type of rat, which is accompanied by an increase in cyclin D1 and p21 expressions.

209-214

Authors: Shaghayegh Anvari , Shahin Najar Peerayeh , Mehrdad Behmanesh , Mina Boustanshenas

Objective: Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein. Materials and Methods: In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). Results: The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 μg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. Conclusion: This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.

225-230

Authors: Bahar Movaghar , Saeedeh Askarian

Objective: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. Materials and Methods: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test). Results: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). Conclusion: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate.

203-212

Authors: Laleh Nikfarjam , Parvaneh Farzaneh

One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture.

223-228

Authors: Zohreh Ahangari , Mandana Naseri , Maryam Jalili , Yasaman Mansouri , Fatemeh Mashhadiabbas , AnahitaTorkaman

Objective: Evaluation of the effect of Propolis as a bioactive material on quality of dentin and presence of dental pulp stem cells. Materials and Methods: For conducting this experimental split-mouth study,a total of 48 maxillary and mandibular incisors of male guinea pigs were randomly divided into an experimental Propolis group and a control calcium hydroxide group. Cutting the crowns and using Propolis or calcium hydroxide to cap the pulp, all of the cavities were sealed. Sections of the teeth were obtained after sacrificing 4 guinea pigs from each group on the 10th, 15th and 30th day. After they had been stained by hematoxylin and eosin (H&E), specimens underwent a histological evaluation under a light microscope for identification of the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of the material used. The immunohistochemistry (IHC) method using CD29 and CD146 was performed to evaluate the presence of stem cells and the results were statistically evaluated by Kruskal-Wallis, Chi Square and Fisher tests. Results: In H&E stained specimens, there was no difference between the two groups in the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of used material(p>0.05). There was a significant difference between the quality of regenerative dentin on the 15th and 30th days (p<0.05): all of the Propolis cases presented tubular dentin while 14% of the calcium hydroxide cases produced porous dentin. There was no significant difference between Propolis and calcium hydroxide in stimulation of dental pulp stem cells (DPSCs). Conclusion: This study which is the first one that documented the stimulation of stem cells by Propolis, provides evidence that this material has advantages over calcium hydroxide as a capping agent in vital pulp therapy. In addition to producing no pulpal inflammation, infection or necrosis this material induces the production of high quality tubular dentin.

237-242

Authors: Masoud Hajia , Ali Amirzargar , Mehdi Ghoreishi , Sohrab Sam

Objective: A reduction in new human immunodeficiency virus(HIV) cases is one of the ten areas prioritized by the United Nations Program on HIV. However, recent official reports confirm the HIV rate is increasing and predicted a huge incidence in the near future in Iran, despite the preventative program by Iran’s Health Ministry. In this descriptive study, we evaluate the frequency of HIV positive cases among referral patients to a private clinic laboratory for its diagnosis in addition to specimens from other laboratories. An epidemiological analysis is also performed. Materials and Methods: In this descriptive study, the total number of patients was 138 cases that referred for the diagnosis of HIV to the private Laboratory. Of these, 93 males (67.4%) and 45 females (32.6%) voluntarily requested to be examined for specific increases in specific antibody titer, western blot assays and RNA quantitation polymerase chain reaction. We collected two separate tubes of whole blood, one for reverse transcriptasepolymerase chain reaction analysis and the second one for the remaining two tests. Those patients who were antibody positive by western blot and/or reverse transcriptase-polymerase chain reaction(RT-PCR) analyses were considered as HIV positive cases. Results: There were 18.84% confirmed HIVcases (17.39% males; 1.45% females). Analysis of the results confirmed that the ratio of male to female patients in the infected group was not comparable to those in the suspect group. The majority of HIV positive cases were either infected by their partner via sexual intercourse (84.61%) or needle sticks (11.53%) among the drug addicted group. The infection routes of the remainder were unknown. Conclusion: Analysis of the data revealed a higher frequency of HIVin males than females among the tested group. There was a shift in to unsafe sexual intercourse as seen in the present study. The higher rate of infected male patients shows a shift in transmission route to unsafe intercourse. Therefore, it is necessary to design new supportive programs by actively identifying and contacting at-risk groups, particularly infected females who are uninterested in being and monitored.

251-258

Authors: Mitra Khalili , Majid Sadeghizadeh , Kamran Alimoghaddam , Reza Malekzadeh , Mohammad Vasei , Seyed Javad Mowla

Objective: microRNAs (miRNAs) are a new class of non-coding RNAs involved in regulating various biological processes including proliferation, differentiation, and apoptosis, among others. Alterations in miRNA expression are reported in several human cancers, which suggests their potential roles in tumor initiation and progression. Members of the miR-302 cluster are highly expressed in embryonic stem cells (ESC), where they regulate cell self-renewal and pluripotency. Based on the cancer stem cell (CSC) hypothesis, mis-expression of such genes might contribute to tumorigenicity. This study aims to find a potential link between the expression level of human/homo sapiens miR-302b (has-miR- 302b) and tumor/grade state of gastric tissues. Materials and Methods: A matched based case-control study was conducted that included tumor and matched marginal non-tumor surgical specimens from 34 patients diagnosed with gastric adenocarcinoma. Randomly selected samples were obtained from the Iran National Tumor Bank. cDNA synthesis was carried out on total RNA, by using the miRCURY LNA™ Universal RT microRNA PCR Kit. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assays were performed with specific LNA™ primers and SYBR Green master mix. The human embryonic carcinoma cell line, NTERA2 (NT2) and a human gastric adenocarcinoma cell line, AGS, were used to optimize the PCR reactions. A comparative evaluation of miR-302b expression in tumor and non-tumor gastric samples was performed by either paired t test or Wilcoxon non-parametric test. The ability of miR-302b to discriminate tumor from non-tumor gastric samples was evaluated using the area under the receiver operating characteristic (ROC) curve. Results: According to our data, miR-302b expression (normalized to that of the U6 snRNA housekeeping gene) in the pluripotent cell line NT2 was more than 500 times greater than that of the AGS cell line. The level of expression was even lower in tumor and non-tumor gastric tissue samples. The data further revealed a down-regulation of miR-302b in gastric tumor samples (p=0.001), particularly in high-grade adenocarcinoma (p=0.009). However, ROC analysis data demonstrated a low sensitivity and specificity of miR-302b expression to discriminate between the tumor and non-tumor state of the samples (AUC=0.63). Conclusion: Despite the upregulation of some hESC-specific genes in tumors, our data revealed a down-regulation of miR-302b in high-grade tumors. This data suggested a potential tumor-suppressor role for miR-302b in tumorigenesis of gastric tissue.

265-274

Authors: Jafar Alijan-pour , Kataneh Abrari , Taghi Lashkar bluki , Mohammad Taghi Ghorbanian , Iran Goudarzi , Mahmoud Elahdadi Salmani

Objective: This research study is an attempt to examine whether the administration of ethanol after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this administration alters the density of Cornu Ammonis (CA)1 and CA3 pyramidal and dentate gyrus (DG) granule cells. Materials and Methods: In this experimental study, adult male Wistar rats (200-300 g) were trained in a fear conditioning system using two 1 second, 0.6 mA shocks with an interval of 180 seconds. Twenty four hours later rats were returned to the chamber for 120 seconds. Immediately after reactivation they were injected with ethanol (0.5, 1, 1.5 mg/ kg) or saline. 1, 7 and 14 days after reactivation, rats were returned to the context for 5 minutes. Seconds of freezing (absence of all movement except respiration) were scored. In the second experiment (described in the previous paragraph), after test 1, animals were anesthetized with sodium pentobarbital and perfused transcardially with phosphate buffer (10 minutes) and 4% paraformaldehyde (15 minutes). The brains were postfixed in phosphate-buffered 4% paraformaldehyde (24 hours) and 30% sucrose. 10-μm sections were stained with cresyl violet. Data were analyzed by 1-and 2-way ANOVA for repeated measurements by means of SPSS 16.0. Tukey’s post hoc test was performed to determine the source of detected significant differences. P <0 .05 were considered significant. Data are presented as mean ± SEM. Results: Findings from the first experiment indicated that ethanol at a dose of 1.5 mg/kg significantly impaired recall of memory only in the first test. The density of CA1 and CA3 pyramidal and DG granule cells in the ethanol group was decreased (p< 0.01) compared with control group respectively 43.7%, 35.8%, and 37.8. Conclusion: The data demonstrate that ethanol exposure impairs post retrieval processes. Moreover, ethanol decreases the density of CA1, CA3 and DG cells. Presumably it would be a correlation between our behavioral and histological results.

281-289

Authors: Shahram Seyedi , Alireza Andalib , Abbas Rezaei , Seyed Mohsen Hosseini , Seyed Reza Mohebbi , Mohammad Reza Zali , Mohamad Vafai , Roubik Behboo , Seyed Abbas Tabatabaei , Shahram Shahabi

Objective: Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed. Materials and Methods: In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 μg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 μmol/L of PRO or 1 μmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Results: Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients’ PBMCs and TILs. Conclusion: This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients’ TILs, which might be due to the downregulation of β-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs.