Cell Journal (Yakhteh)
Volume:14 Issue: 2, Summer 2012

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. 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. 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. 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.

We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irradiated testes. In this experimental research, the right testes from adult mice (n=25) were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine (BrdU)-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells (control 1), fresh cells co-cultured with Sertoli cells (control 2), the frozen-thawed cells (experimental 1) and frozen-thawed cells co-cultured with Sertoli cells (experimental 2). The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation. The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient’s testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups (p≤0.001). Our results demonstrated that spermatogonial stem cells in the colonies could result sperm production in the recipient’s testes after autologous transplantation.

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. 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). 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. 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.

Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP). Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNALys genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis. In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments. In this study, 12 patients (50%) showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions (55.55%) were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis. MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions.

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. 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. 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. 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.

In electrochemotherapy (ECT), there is an unpleasant sensation of muscle contraction when using a low frequency (1 Hz). Therefore, by increasing the pulse frequency above the tetanic frequency this painful sensation can be reduced. The aim of the present study is to compare the treatment efficiencies of low and high frequency ECT, and estimate the effect of its repeated sessions. We transplanted invasive ductal carcinoma into the flanks of female Balb/c mice. ECT was performed on the mice by the use of 8 pulses, 1000 v/cm, of 100 μs duration at 1 Hz and 5 kHz repetition frequencies along with intra-tumoral injections of bleomycin. We also used this ECT protocol for the second therapy session six days after tumour regrowth. The effect of treatment was measured by calculating the tumor volumes for 24 days following treatment. Statistical analysis was performed with ANOVA. ECT at 1 Hz and 5 kHz pulse frequencies demonstrated significant inhibition of tumor growth, but after the first treatment the tumours began to regrow. Repetitive ECT sessions increased the curability of tumors up to 40% in the group treated by 1 Hz frequency and 60% in the group treated with 5 kHz frequency. Our results demonstrate that the effects of 1 Hz and 5 kHz pulse repetition frequencies are comparable for inhibited tumour growth. Repetitive treatment can improve the effectiveness of ECT.

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). 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. 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. American ginseng can prevent the cytotoxic effects of CP on sperm quality factors.

In previous studies it has been emphasized that the site of morphine action may be either in the embryo or the placenta. In the present study, we attempt to identify the site of morphine action on the fetal section of Wistar rat placenta by using C14-morphine. In this study (experimental), female Wistar rats (weights: 170-200 g) were mated with male rats and their coupling times recorded. Experimental groups received daily doses of 0.05 mg/ml of C14-morphine in their drinking water. On the 9th and14th embryonic days, the pregnant rats were anesthetized and the placenta and uterus surgically removed. Placentas were fixed in 10% formalin for two weeks, then processed, sectioned in 5 μm and 25 μm thicknesses, and fixed on glass slides for further evaluation. The 25 μm sections were delivered to black and white film for three days. Films were processed and evaluated with a digital inverse microscope for possible radiological impression. The 5 μm sections were processed for hematoxylin and eosin (H&E) staining, and evaluated by light microscope and MOTIC software. Our results indicated that the site of action of C14-morphine was possibly located on the blood plexus of the fetal portion of the placenta. In addition, oral morphine consumption was shown to inhibit fetal and maternal placental development in the experimental groups. We conclude that morphine’s effectiveness on the reduction of embryo growth and development may be via its effects on the blood plexus of the fetal section of the placenta.

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. 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. 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. 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.

The aim of this study was to test the effect of intravenous injection of mesenchymal stem cells (MSCs) on doxorubicin (DOX)-induced fibrosis in the heart. We investigated the mechanisms that possibly mediate this effect. In this experimental study, fibrosis in the myocardium of adult male Wistar rats (weights 180-200 g, 9-10 weeks of age, total n=30) was created by DOX administration. DOX (2.5 mg/kg) was administered intraperitoneally 3 times a week, for a total dose of 15 mg/kg over a period of 2 weeks. MSCs from Wistar rats were separated and cultured in Dulbecco’s modified eagle medium (DMEM). The condition medium (CM) which contained factors secreted by MSCs was also collected from MSCs cultured in serum-free DMEM. Two weeks after the first injection of DOX, MSCs, CM and standard medium (SM) were transplanted via intravenous injection. Four weeks after transplantation, histological (Masson’s trichrome staining for fibrosis detection) and molecular [real-time polymerase chain reaction (RT-PCR)] analyses were conducted. In addition, insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF) in the CM were measured with an enzyme-linked immunosorbent assay (ELISA). For immunosuppressive treatment, cyclosporine A was given (intraperitoneally, 5 mg/kg/day) starting on the day of surgery until the end of study in all groups. Fibrosis rate and relative gene expression were compared by analysis of variance (ANOVA) and post-Tukey’s test. HGF and (IGF-1 in the CM were analyzed by independent sample t test. P<0.01 was considered statistically significant. Our data demonstrated that intravenously transplanted MSCs and CM significantly reduced fibrosis and significantly increased Bcl-2 expression levels in the myocardium compared to the DOX group (p<0.01). However, there was no significant difference between Bax expression levels in these groups. In addition, secretion of HGF and IGF-1 was detected in the CM (p<0.01). We conclude that intravenous transplantation of MSCs and CM can attenuate myocardial fibrosis and increase Bcl-2 expression. This may be mediated by paracrine signaling from MSCs via anti-fibrotic and anti-apoptotic factors such as HGF and IGF-1.