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Background: Cerebral venous sinus thrombosis is a rare form of brain stroke caused by thrombosis in venous sinuses of Dura. This study reports on a patient with venous sinus thrombosis and brucellosis who presented with uncontrolled seizure. Case: A 33-year-old woman with past history of controlled complex partial seizure who presented with headache, asthenia, and uncontrolled seizure for one month is described in this study. She was febrile and in brain CT scan hemorrhagic focus in left posterioparietal and temporal lobe was reported. MRI and MRV proved venous sinus thrombosis in left transverse sinus. In laboratory assessment, brucellosis was confirmed as well. The patient treated with anticoagulant, anti-brucellosis, and antiepileptic agents and discharged in good condition with medication orders. Conclusion: Clinical suspicion and accurate evaluation is the most important clue in the diagnosis and treatment of brucellosis and venous sinus thrombosis, especially in uncontrolled seizure in patients who had previously been under control.

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Background: The importance of VP2 protein of canine parvovirus to bind to human cancer cells and to detect the virus in veterinary detection kits has motivated a lot of research on the production of this protein. In this project, a surface gene of canine parvovirus (VP2) was cloned and expressed in a prokaryotic vector system and its expression was optimized in a specific cell-free prokaryotic expression system. Materials and Methods: In this experimental study, plasmid pET-21aVP2 was constructed by cloning the PCR product of VP2 gene of canine parvovirus into the plasmid expression pET-21a vector. The best sequence was analyzed through PCR and it was followed by confirmation with sequencing and restriction digestion. To produce VP2 protein, plasmid pET-21aVP2 was transferred into Escherichia coli, Rosetta (DE3) strain, and the expression of this protein was induced by IPTG. The production of VP2 protein in both systems was evaluated using SDS PAGE technique. The expressed protein was checked with monoclonal antibody against VP2 protein by Western blotting technique. Results: Successful cloning of VP2 protein was confirmed by enzymatic digestion and sequencing. The expression of VP2 protein in bacterial and cell-free prokaryotic systems was verified by SDS PAGE and the specific band in Western blotting also confirmed the VP2 protein. Conclusion: The results of this study showed that VP2 gene was amplified in the cloning phases and it was successfully cloned in the expression vector. Protein expression was confirmed in both bacterial and cell-free prokaryotic systems.

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Background: Brucellosis is a debilitative disease that imposes heavy costs on the economy and society. Therefore, using the most accurate and efficient method to diagnose this disease is essential. In Iran, Brucella melitensis is the common causative agent for brucellosis and BP26 protein of this bacterium has a good level of antigenicity. Thus, the aim of this study is to produce Brucella melitensis recombinant BP26 protein with a PET28a expression vector.

Materials and Methods: In this applied-fundamental study, genomic DNA was isolated from bacterial culture through proteinase K (pK) and phenol/chlorophorm protocol. Then, two pairs of primers were designed based on the known sequence in the gene bank for amplification of Brucella melitensis bp26 gene and PCR reaction was set up and optimized. The PCR product was cloned first into PTZ57R/T vector and accessed on the PET28a vector and sequenced. The recombinant vector was transformed and expressed into E. coli BL21 (DE3). Then, the recombinant protein was purified with Ni-NTA column of chromatography against His tag.

Results: The size of PCR product was in accordance with the part of bp26 gene size in the gene bank. The bp26 gene without adding IPTG had little expression and 3 hours after adding IPTG with a 1 Mm concentration to culture media, extreme expression was observed.

Conclusion: The production of recombinant BP26 protein from isolated Brucella melitensis native to Markazi province was done. Noticing the importance of BP26 protein and its significance for future studies on providing brucellosis diagnosis kits, its production was made possible.

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Background: Retinoic acid (RA) is a vitamin A derivative and one of the most important inducing signals in vertebrates that is involved in differentiation, morphogenesis, apoptosis, and reproduction. This study was done to evaluate the role of RA in in vitro neural patterning of mouse embryonic stem cells (mESCs).

Materials and Methods: In this experimental study, upon formation, embryoid bodies (EBs) from mESCs, Royan B1, were induced by 1 µM RA for four days and then plated for eight days. Untreated EBs were considered as the control group. Finally, in both groups, neural induction and patterning of EB-derived neural cells were evaluated by using immunostaining, flowcytometry, and RT-PCR methods.

Results: RA induced neurogenesis in ES cells, from which 35% showed to express MAP2. RT-PCR analysis also indicated that RA-treated neural cells derived from ES cells could at the same time express Mash1, Pax6/7, and Dbx1/2 as dorso-ventral (DV) pattering markers and Hoxb4, Hoxc5, and Hoxc8 as the rostro-caudal (RC) axis markers.

Conclusion: RA induces in vitro neural induction along with neural patterning of ES-derived neural cells in DV and RC axes. Keywords: Mouse embryonic stem cells, neural patterning, retinoic acid

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Background: The present study aimed to investigate the immunomodulatory activity of molecules secreted by decidual cells on dendritic cells (DCs) function in abortion-prone compared with non-abortion-prone mice.

Materials and Methods: The decidual cell supernatants (DS) were obtained from abortion and non-abortion mouse models. DCs were purified from CBA/J mice spleens and treated with antigen and DS. Treated DCs were injected into mice palms. After 5 days, draining lymph nodes were removed, cultured in the presence of cognate antigen, and proliferation of lymphocyte cells was measured by 3H-thymidin incorporation.

Results: Our results showed that immunosuppressive activity of DS from non-abortion-prone mice significantly decrease dendritic cells' ability to stimulate lymphocytes proliferation compared with DS from abortion-prone mice (Simulation index (SI) of 4.93 ± 0.34 versus 11.84 ± 0.79).We, also found that DS prepared from non-resorption sites compared with DS from resorption sites in abortion-prone mice had increased immunosuppressive activity on DC function (SI of 7.31 ± 1.02 versus 2.67 ± 0.49).

Conclusion: Due to our results, we concluded that immunomodulatory activity of molecules secreted within decidual tissue is different between abortion-prone and non-abortion-prone mice. Based on the key role of DCs in inducing fetomaternal tolerance, we claimed that these molecules, through modulation of DCs function, play crucial role on pregnancy outcome.

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Background: All-trans retinoic acid(RA), an active metabolite of vitamin A, is widely used to induce cell differentiation. It has significant effects on growth and proliferation of epithelial cells. It also causes cell cycle arrest in G0/G1 phase and induces the apoptosis. cisplatin, a chemotherapy compound that cross-linking to DNA, and leads to apoptosis, it is commonly used for treatment of ovarian, head and neck, esophageal, gastric cancers and melanoma. Recent studies showed that RA enhances cytotoxic effects of cisplatin on melanoma and ovarian cancer. Our literature review showed that there is no previous study on the effect of RA in combination with cisplatin on esophageal cancer, hence current study conducted to investigate such combination treatment on esophageal derived cell, KYSE-30.

Materials and Methods: KYSE30 cell line was cultured in presence of different concentration of RA alone and in combination with cisplatin. Then, cell death was investigated by colonogenic assay and acridine orange/ ethedium bromide staining.

Results: The results showed that RA concentrations &ge15µM cause differentiation of KYSE30 to squamous cell morphology, while lower concentrations decreases the colony formation (p&le0.05). These effects were also observed in combination with cisplatin and RA. The best effects on cell death were observed in 10 µM of RA of combination with 5 and 10 µg/ml of cisplatin.

Conclusion: The results suggest that low concentration of RA in combination with cisplatin are more effective than cisplatin alone in terms of apoptosis and necrosis of esophageal cancer, KYSE-30.

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Background: Wharton's Jelly Mesenchymal Stem Cells (WJMSc) are potential renewable source of cells in replacement therapies of many diseases. Different biomaterials have been used as a scaffold to mimic the stem cell niche, which is important for promoting cellular interactions, cell proliferation and differentiation. Encapsulation involves entrapment of living cells within the semi-permeable membrane for the exchange of nutrients, oxygen and stimuli, whereas antibodies and host immune cells are kept out. In this study, a new approach for culturing and differentiating Wharton's Jelly Mesenchymal Stem Cells to definitive endoderm in a three dimensional environment using alginate capsules is presented.

Materials and Methods: In this experimental study, Wharton's Jelly Mesenchymal Stem Cells were capsulated and Trypan blue exclusion method was applied to determine cells viability. Then, encapsulated cells have been cultured in medium contain differentiating factors and to investigate the expression of definitive endoderm related genes, Real- time PCR was performed.

Results: The encapsulation procedure did not alter the morphology and viability of the encapsulated cells. Post-differentiation analysis confirmed the expression of FOXa2 and SOX17 as definitive endoderm specific markers.

Conclusion: Alginate has potential to be used as a three dimensional scaffold for culturing and differentiation of WJMSCs to definitive endoderm.

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Background: Treatment failure and relapse is a major problem in the treatment and control of brucellosis. The present study directed to determine risk factors for failure of treatment and relapse in patients treated for brucellosis.

Materials and Methods: This study was a descriptive - analytic and prospective study and were conducted in 72 patients with acute brucellosis.Patients were followed up during treatment and for six months after completion of therapy, and patients with treatment failure or relapse were analyzed. All data analyzed using SPSS software and P-value of less than 0.05 was considered significant.

Results: The mean value of age in patients was 40.2 ± 16.5 years. Treatment failures was 6.9% and the relapse at the end of the ninth month was 12.5% that recurrence of symptoms were associated with increased titers of serological tests and 80.6 percent had successful treatment. In this study gender of male (P = 0.026), occupational exposure (P = 0.005), delayed treatment in less than two weeks (P = 0.016), hepatosplenomegaly (P = 0.003), thrombocytopenia (P = 0.023), CRP &ge +2 (P = 0.017) and Wright &ge 1/320 and 2-ME &ge 1.160 at the end of the sixth week of treatment (P = 0.004 and P = 0.010) were risk factors of treatment failure and relapse in brucellosis.

Conclusion: The diagnosis and treatment of relapse and treatment failure in acute brucellosis is not clear, therefore, the prediction of relapse may be useful in preventing recurrence and treatment of patients.

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Background: Brucellosis is a major cause of zoonosis disease and is endemic in hamadan Province in Iran. The purpose of this study was to isolate Brucella species from brucellosis patients and identify different species of this bacterium in order to determine the prevalence of the species.

Materials and Methods: This study was descriptive- cross sectional and fifty blood samples were obtained from brucellosis patients with clinical symptoms. The samples were cultured in BACTEC system and incubated for 14 days. Then, the samples were cultured on Brucella agar and biochemical tests were done for identification of bacteria. Finally, Polymerase Chain Reaction (PCR) applied for confirmation and isolated identification with specific primers.

Results: Seven Brucella strains were isolated from 50 blood samples of the patients with brucellosis by blood culture and PCR. The PCR results on blood specimens showed 4 positive in spite of the negative results of blood culture. PCR and biochemical methods revealed that all the 11 isolated bacteria were Brucella Melitensis.

Conclusion: This study was designed to evaluate PCR technique as a diagnostic tool for brucella spp in comparison to conventional techniques. This study showed a high prevalence of brucellosis due to Brucella Melitensis in Hamadan Province and efforts in this region should be aimed at the eradication of this bacterium.

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Background: Cancer stem cells are subpopulation of cancer cells that show self-renewal potential and the capacity to differentiate into diverse populations comprising a tumor. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free media. The aim of this study was isolation of colon cancer stem cells by sphere formation assay and characterization of them in human colonic adenocarcinoma HT-29.

Materials and Methods: In this experimental study, colon CSCs markers including CD44 and EPCAM in spheroid and HT-29 cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and HT-29 cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated using in vivo xenografts assay.

Results: Our data showed over 92% of spheroids were CD44+/EpCAM+, while HT-29 cells only have expressed 37% of CD44/EpCAM markers. In compared with the HT-29 cells, expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells (p< 0.05). Further, As little as 2500 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x106 of HT-29 cells was needed to form tumor.

Conclusion: Our data suggest that spheroid formed by colon cancer cell lines highly enriched in CSCs and showed increasing expression of stemness genes and tumorigenic in nude mice.

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Background: Inflammation has an important role in the pathogenesis of atherosclerosis. The aim of this study was to evaluate the role of WBC (White Blood Cell) count and incidence of morbidity and mortality in patients with ACS.

Materials and Methods: This prospective study was carried out on 101 patients with ACS who admitted in Bu-ali Sina hospital. All patients were stratified according to WBC categories in to 3 groups, (WBC 1 :<7000 mm3, WBC 2:7000 -10000 mm3, WBC 3:> 10000 mm3). Demographic and laboratory data such as acute reactive protein, cardiac biomarker and etc. were recorded. Adverse cardiac events and mortality were recorded to a phone or in person for six months of follow up period. The collected data were analyzed using SPSS software (Statistical Package for Social Sciences, version 17.0). The Fisher´s exact chi-square test and the student t-test were applied. P-values less than 0.05 were considered significant.

Results: In our study, 5 patients (31.25%) in third group had recurrent non fatal cardiac event and the same percent (31.25%) were died after 6 months follow up. Multivariate analysis showed WBC count >10000 mm3 was strongest predictor of outcome in our patients.

Conclusion: WBC count can be considered one of the strong independent predictor of mortality and cardiac event in patients with ACS.

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Background: Hematopoietic stem cell transplants are routinely used to treat patients with cancers and other disorders of blood and immune systems. Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis by participating in the formation of the HSC niche. It is believed that interaction between hematopoietic cells and bone forming osteoblasts regulate each other’s function. It is established that acute blood loss in animal models activates bone formation and niche development because of EPO stimulation. In this experimental study we have examined the co-culture of HSCs derived from cord blood which treated with EPO in vitro, on osteoblastic differentiation of mesenchymal stem cells.

Materials and Methods: In this experimental study MSCs isolated from bone marrow and co-cultured with CD 34+ CD38- HSCs isolated from cord blood. These co-cultured cells were treated with different doses of erythropoietin for 14 days, after that RNA were extracted from MSCs and analysed with RT-PCR to evaluate the expression of osteopontin and osteocalcin. Alizarin red and alkaline phosphatase staining were done for osteoblastic differentiation.

Results: Osteopontin and osteocalcin were expressed in MSCs. Cellular staining were positive for osteoblastic differentiation. Differentiated cells expressed osteoblastic markers.

Conclusion: These data suggest that EPO regulates the osteoblastic differentiation from bone marrow MSCs in vitro.

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Background: Delivery of antigens directly to dendritic cells enhances the immune responses to the antigen and is an attractive approach for eliciting cellular immune responses against mutagenic pathogens like HIV virus. So the aim of this study is evaluation of immune responses elicited by delivered multi-epitopic HIV-1 tat/pol/gag/env recombinant protein to dendritic cells in sito using &alphaDEC-205 mAb.

Materials and Methods: In this study, recombinant protein expressed by pET23a-HIVtop4 plasmid including HIVtop4 sequence (Gag158-186, Pol150-190, ENV296-323, ENV577-610, Tat1-20 and Tat44-61) in BL21 E. coli cells was used as vaccine model. To exploiting dendritic cells, properties for immunization purposes, we conjugated this recombinant protein chemically to anti body against DEC-205 receptor on these cells. Balb/c mice immunized subcutaneously (s.c.) with conjugated multi-epitopic protein or un-conjugated one (as control) simultaneously with Poly I: C as dendritic cell maturation factor. Lymphocyte proliferation was measured by bromo di uridine assay, Cytotoxicity by Grenzyme B production activity, IL-4, IL-17, IFN- cytokines production and total antibody by direct and indirect ELISA methods in order.

Results: Immunization by anti DEC-205 conjugated peptide led to a significant increase in the proliferative responses of lymphocytes, production of Gr-B, IFN-&gamma, IL-4 and IL-17 cytokines and total antibody titer in comparison with the none targeted groups.

Conclusion: It is concluded that targeting of protein antigens to DEC-205+dendritic cells significantly enhances immune responses in compare to non-targeting strategies.

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Background: Nanomaterials have gained increasing attention because of their novel properties, including a large specific surface area and high reaction activity. This study was designed to investigate the cytotoxic effects of CuO nanopaticles on brain, spleen, and embryo NMRI pregnant mice.

Materials and Methods: In this experimental study, forty two female NMRI mice of (weighting 30±3.0 g) were randomly divided into six groups (four experimental groups, one sham group and one control group).The experimental mice on days 3 and 12 of pregnancy received CuO nanoparticle with concentrations 50, 100, 150, 200 mg/kg intraperitoneal injection. On day 17 pregnancy, brain, spleen and fetus weights were measured.Tissues for histopathological evaluation were stained with hematoxylin and eosin.

Results: Based on the macroscopic observations of embryos weight with increasing concentration of nanoparticle compared to control reduces its toxicity increased (p&le0.05). Spleen only at concentration of 600 mg/kg showed significant changes compared to control (p&le0.05). Histopathologic examination on brain and spleen following IP administration of CuO nanoparticle showed signs of cytotoxicity (congestion, necrosis, inflammatory cell infiltration, vacuolar degeneration) and (congestion, necrosis, increased hemosiderin) compared to control group, respectively.

Conclusion: The present study clearly showed that CuO-NPs can produce the histopathological abnormalities on brain and spleen tissues of NMRI mice in a dose-dependent manner.

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Background: The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor of this bacteria. Stimulating the immune system for helicobacter infection treatment could have an important role. The aim of study is to assess the effect of recombinant Neutrophil activating protein (Hp-NapA) of helicobacter pylori on proliferation and viability of peritoneal macrophages from BALB/c mice.

Materials and Methods: In this experimental study, recombinant Hp-NapA of helicobacter pylori was produced in vitro. Mice peritoneal macrophages were purified and cultured. Different concentrations of recombinant Hp-NapA was used for macrophages stimulation. MTT assay was performed to assess the viability and proliferation of macrophages.

Results: The results elucidated that the increasing effect of stimulation with recombinant Hp-NapA was significant at the dose of 30 µg/ml  (p=0.01). The rate of viabitity was significantly higher than control group at the doses of 30 and 60 µg/ml and in the concurrency series of recombinant protein with lipopolysaccharid, there was a statistically significarit increase in proliferation at just these doses.

Conclusion: According to our findings, recombinant Hp-NapA has a positive effect on proliferation, viability and function of peritoneal macrophages. Therefore, it is proposed that recombinant Hp-NapA can be studied as an immunomodulator for immunotherapy.

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Background: Shigella is the causative agent of human shigellosis and its lipopolysaccharide is detected by TLR4. TLR4 belongs to Toll-like receptors family and many immunological pathways are triggered when these receptors are stimulated. Many researches showed increasing in TLR4 expression in mesenchymal stem cells through lipopolysaccharide treatment. The main goal of this study is detecting the optimum lipopolysaccharide between shigella strains through stimulation of immune system for vaccine studies.

Materials and Methods: In this experimental study human bone marrow derived mesenchymal stem cells were treated with three distinct concentrations (0.1, 0.01, and 0.001) of shigella (S. flexneri, S. dysenteriae, S. sonnei) extract containing lipopolysaccharide. Then TLR4 expression in mRNA level was investigated by RT-PCR and Q-PCR. The cells treated with phosphate buffered saline have been considered as a control group.

Results: Expression of TLR4 was shown in all of case groups except treatment with concentration 0.001 of extracts from sonnei and dysenteriae and also control group. The variations in the expression of TLR4 was dose-dependent in all of case groups. The maximum expression of TLR4 related to treatment with extract from shigella flexneri strain and the minimum expression related to treatment with shigella sonnei extract. The use of lipopolysaccharide from E. coli as a positive control indicated that lipopolysaccharide in shigella extracts is responsible for the increased expression of TLR4.

Conclusion: The TLR4 expression level was increasesed by S. flexneri extract, so it could be recommended for increasing vaccine efficiency.

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Background: Brucellosis is a highly contagious zoonotic disease between humans and animals known for high frequency of relapsing and ability to cause chronic infection. The function of immune system plays an important role in induction of chronic diseases. However, the role of immune system response is not completely studied. Therefore, this study designed to investigate the cytokine profile of the patients suffering from chronic and acute brucellosis.

 Materials and Methods: This descriptive- analytical study was performed on 22 patients with acute brucellosis (mean age 38±17), 21 patients with chronic brucellosis (mean age 43±10) and 21 healthy people (mean age 26±4) with the same age and sex as patients. The serum IFN-&gamma, IL-17, IL-5 and TGF- &beta levels were measured using ELISA method.

 Results: The mean IFN-&gamma serum level in acute and chronic brucellosis patients group was significantly higher than control group (p=0.045). The mean IL-17 serum level in acute brucellosis patients was significant decreased once compared with control group and in chronic patients was significantly decreased when compared with control group (p=0.024). In addition, the mean IL-5 and TGF-&beta serum levels of acute brucellosis patients group were significantly decreased as compared to chronic patients (p=0.001).

 Conclusion: The results of current study indicate that cytokine profile of chronic brucellosis patients is more related to Th2 immune response. Hence, Th2 immune response inhibition would be an appropriate way to treat and prevent disease to become chronic.

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Background: Direct transmission of avian influenza viruses with human receptor binding specificity to humans is a serious risk of newly emerging virus responsible for pandemy. The analysis of recent avian influenza hemagglutinin sequences and their glycans show their affinities to the human sialic acid receptors. The upregulation of proinflammatory cytokines and type I IFN genes, and host cell death responses contribute to the pathogenesis of influenza infection. Understanding the host cell-virus interactions and replication dynamic of the viruses in different cells is an essential step in surveillance and controlling programs against influenza.

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Background: Wound healing is a complex process that is impaired in diabetic patients due to several factors. So far, the positive effects of mesenchymal stem cells secretions in wound healing process have been reported. In this study, we investigated the effect of human mesenchymal stem cells Conditioned media on expression of effective factors involved in wound healing.

Materials and Methods: 27 rats were divided into 5 groups: no wound control, normal control, diabetic control, diabetic placebo and diabetic experimental. Diabetes was induced by Alloxan. A wound was created on the back of the rats. Then, the conditioned medium was prepared from mesenchymal stem cells. Diabetic experimental rats received 200 microliter of conditioned medium intravenously. The wounds were sampled and expression of KGF and TGF-&beta1 genes was examined by RT-PCR on days four and seven after wounding.

Results: In the diabetic experimental group, expression of KGF gene at fourth and seventh days had been non-significantly increased in comparison to diabetic control group. While, expression of TGF-&beta1 gene in diabetic experimental group compared to diabetic control group had been significantly (p<0.05) increased on fourth day, and non-significantly increased on seventh day.

Conclusion: It seems that using the conditioned medium derived from human mesenchymal stem cells positively affects the expression of trophic and inflammatory factors involved in diabetic skin wound healing.

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  Background: Alpha-tocopherol, as a strong antioxidant, plays an important role in testraining free radicals. The aim of this study was to investigate the effect of Alpha-tocopherol on cell proliferation and restraining apoptosis in rat bone marrow mesenchymal stem cells.

  Materials and Methods: In this research study, the rat bone marrow mesenchymal stem cells were extracted under sterile conditions using flashing-out method. At the end of the third passage, cells were divided into groups of control and Alpha-tocopherol with doses of 15 and 25 µM and were treated in the osteogenic media cell medium containing 10% fetal bovine serum, 10 mM β-glycerol phosphate, 10 nM dexamethasone and 50 µg/ml ascorbic 3-phosphate] for a period of 21 days. Then, cell proliferation, DNA damage, expression of Bcl-2 and Bax genes and the morphologic changes of the cells were investigated during the procedure of osteogenesis. Data were analyzed using one-way ANOVA and means difference was considered significant at p<0.05.

  Results: Cell proliferation, the size of nuclei diameter and expression of anti-apoptotic Bcl-2 gene showed a significant increase in mesenchymel stem cells treated with Alpha-tocopherol (p<0.05) in a dose dependent manner compared to the control cells. Also, cytoplasm extension was seen in the cells treated with Alpha-tocopherol, compared to the control group. Since Alpha-tocopherol causes a significant decrease in DNA damage and the expression of apoptotic Bax gene, compared to the control group, therefore it can suppress apoptosis in bone marrow mesenchymal stem cells, in a dose dependent manner .