Effect of Different Activation Mechanism of T Cells on Immunoregulatory Properties of Bone Marrow Mesenchymal Stem Cells

Background: Mesenchymal Stem Cells (MSC) have ability to regulate immune response via proinflamatory cytokines. In this study we aimed to investigate the effect of different mechanism for T cell activation and adjacency on immunoregulatory properties of MSC.Methods: Bone marrow MSCs were purchased and T cells were obtained from whole blood sample. T cells were activated by two different mechanisms using Peripheral Blood Mononuclear Cell (PBMC) and phytohemagglutinin (PHA) and then co-cultured with MSC using transwell cultures (Indirect contact) and usual plate (Direct contact). After 96 hours TGF-b concentration in culture medium and indole amine 2, 3-dioxygenase (IDO) activity in cell lysate were assayed. We used ANOVA and T-test for statistical analysis and 0.05 was considered as significant level.Results: Our result showed a significant increase of TGF-b secretion and IDO activity. Increase in mixed lymphocyte culture (MLC) groups was more significant than lymphocyte transformation test (LTT) in compare with control.Conclusions: The present study confirmed immunoregulatory effect of BM-MSC. Also this study showed that MLC of BM-MSCs and T cells have more immunoregulatory effects than LTT.

Effect of Different Activation Mechanism of T Cells on Immunoregulatory Properties of Bone Marrow Mesenchymal Stem Cells

Background: Mesenchymal Stem Cells (MSC) have ability to regulate immune response via proinflamatory cytokines. In this study we aimed to investigate the effect of different mechanism for T cell activation and adjacency on immunoregulatory properties of MSC.Methods: Bone marrow MSCs were purchased and T cells were obtained from whole blood sample. T cells were activated by two different mechanisms using Peripheral Blood Mononuclear Cell (PBMC) and phytohemagglutinin (PHA) and then co-cultured with MSC using transwell cultures (Indirect contact) and usual plate (Direct contact). After 96 hours TGF-b concentration in culture medium and indole amine 2, 3-dioxygenase (IDO) activity in cell lysate were assayed. We used ANOVA and T-test for statistical analysis and 0.05 was considered as significant level.Results: Our result showed a significant increase of TGF-b secretion and IDO activity. Increase in mixed lymphocyte culture (MLC) groups was more significant than lymphocyte transformation test (LTT) in compare with control.Conclusions: The present study confirmed immunoregulatory effect of BM-MSC. Also this study showed that MLC of BM-MSCs and T cells have more immunoregulatory effects than LTT.

Effect of Thymus Vulgaris Ethanol Extract, on Serum Total Antioxidant in Experimental Induced Poly Cystic Ovarian Syndrome (PCOs) Rats

Background: Poly cystic ovary syndrome is one of the most common female endocrine disorders. One of the side effects of PCOS is oxidative stress.. Here we investigated antioxidants effects of Thymus vulgaris ethanoli extract on experimental PCOS induced rats by estradiol-valerat (PPA).Methods: Wistar female rat (n=70) were divided into 7 groups including C1: an equal volume of (0.9% NaCl) as placebo; C2: extract (0.6cc/rat/orally/daily); C3: induced PCO by single injection of estradiol-valerate (4mg/rat/IM), and T1: PCOS induced  rats + an equal volume of (0.9% NaCl) as placebo, T2: PCOS induced rats + extract(0.2cc/rat/orally/daily), T3: PCOS induced rats + extract (0.4cc/rat/orally/daily), T4:PCOS induced rats+extract (0.4cc/rat/orally/daily) test groups, were received extract supplement, for 60 consequence days. Animals were kept in standard conditions. In last day of study the blood samples of rats in whole groups were obtained and prepared to biochemical analysis.Results: Total antioxidant capacity level, superoxide dismutase and catalase activity were significantly increased in PCOS treated groups (P<0.03), these parameters in PCOS groups that did not receive extract significantly decreased (P<0.05) in comparison to control. Level of MDA in PCOS groups were significantly increased as compared to control and extract treated groups (P<0.01).Conclusions: Our results disclosed that administration of Thymus vulgaris ethanol extract significantly restitution tissue antioxidants level in PCOS induced rats.

Effect of Thymus Vulgaris Ethanol Extract, on Serum Total Antioxidant in Experimental Induced Poly Cystic Ovarian Syndrome (PCOs) Rats

Background: Poly cystic ovary syndrome is one of the most common female endocrine disorders. One of the side effects of PCOS is oxidative stress.. Here we investigated antioxidants effects of Thymus vulgaris ethanoli extract on experimental PCOS induced rats by estradiol-valerat (PPA).Methods: Wistar female rat (n=70) were divided into 7 groups including C1: an equal volume of (0.9% NaCl) as placebo; C2: extract (0.6cc/rat/orally/daily); C3: induced PCO by single injection of estradiol-valerate (4mg/rat/IM), and T1: PCOS induced  rats + an equal volume of (0.9% NaCl) as placebo, T2: PCOS induced rats + extract(0.2cc/rat/orally/daily), T3: PCOS induced rats + extract (0.4cc/rat/orally/daily), T4:PCOS induced rats+extract (0.4cc/rat/orally/daily) test groups, were received extract supplement, for 60 consequence days. Animals were kept in standard conditions. In last day of study the blood samples of rats in whole groups were obtained and prepared to biochemical analysis.Results: Total antioxidant capacity level, superoxide dismutase and catalase activity were significantly increased in PCOS treated groups (P<0.03), these parameters in PCOS groups that did not receive extract significantly decreased (P<0.05) in comparison to control. Level of MDA in PCOS groups were significantly increased as compared to control and extract treated groups (P<0.01).Conclusions: Our results disclosed that administration of Thymus vulgaris ethanol extract significantly restitution tissue antioxidants level in PCOS induced rats.

Cloning and expression of human vasohibin1 gene in E. coli

Background: Angiogenesis is an important process in various physiologic and pathologic states. The most significant stimulator of angiogenesis is vascular endothelial growth factor (VEGF). In contrast, vasohibin1 acts as an angiogenesis inhibitor which specifically inhibits new vessels formation. The aim of the present study was cloning and expression of vasohibin1 gene in E. coli as well as purification of recombinant vasohibin1 protein. Methods: Total RNA was extracted from human umbilical vein endothelial cells and cDNA was synthesized by RT-PCR. cDNA was amplified using a specific designed primer set. The PCR product was evaluated by electrophoresis and then cloned in pET28a expression vector which transformed into E.coli BL21 (DE3) as a host. IPTG is used as an expression inducer in media. Alternatively, PCR products were analyzed by sequencing and double digestion with EcoRI and HindIII restriction endonuclease. The expressed protein was purified by Ni-NTA column and confirmed by SDS Page and western blotting. Evaluation of gene inhibition was carried out through western blottting and RT-PCR. Results: No mutation or sequence variants were found in PCR products as a result of sequencing analysis. Moreover, the quantity and quality of expressed recombinant protein in the presence of IPTG with selected vector in E. coli was approximately high. VASH1 significantly prevented the receptor expression. The quality and level of expressed protein in pET28 expression vector indicated the efficacy of the applied system in vasohibin1 production. Conclusions: The produced vasohibin1 protein probably can be used as an angiogenesis inhibitor in further studies on retinopathies

Cloning and expression of human vasohibin1 gene in E. coli

Background: Angiogenesis is an important process in various physiologic and pathologic states. The most significant stimulator of angiogenesis is vascular endothelial growth factor (VEGF). In contrast, vasohibin1 acts as an angiogenesis inhibitor which specifically inhibits new vessels formation. The aim of the present study was cloning and expression of vasohibin1 gene in E. coli as well as purification of recombinant vasohibin1 protein. Methods: Total RNA was extracted from human umbilical vein endothelial cells and cDNA was synthesized by RT-PCR. cDNA was amplified using a specific designed primer set. The PCR product was evaluated by electrophoresis and then cloned in pET28a expression vector which transformed into E.coli BL21 (DE3) as a host. IPTG is used as an expression inducer in media. Alternatively, PCR products were analyzed by sequencing and double digestion with EcoRI and HindIII restriction endonuclease. The expressed protein was purified by Ni-NTA column and confirmed by SDS Page and western blotting. Evaluation of gene inhibition was carried out through western blottting and RT-PCR. Results: No mutation or sequence variants were found in PCR products as a result of sequencing analysis. Moreover, the quantity and quality of expressed recombinant protein in the presence of IPTG with selected vector in E. coli was approximately high. VASH1 significantly prevented the receptor expression. The quality and level of expressed protein in pET28 expression vector indicated the efficacy of the applied system in vasohibin1 production. Conclusions: The produced vasohibin1 protein probably can be used as an angiogenesis inhibitor in further studies on retinopathies