| Mesenchymal stem cells (MSCs) are multipotent differentiation potential of adult stem cells to evade the host immune system surveillance, immune modulation and in vitro culture and easy expansion characteristics, so that bone marrow mesenchymal stem cells in cell therapy particularly with applications. However, the death of transplanted cells limited tissue regeneration. one of the death mechanisms of Stem cells transplanted into the organ caused by ischemia.Hypoxic / ischemic conditions, as apoptosis is an important regulator of the media and has been recognized early. This situation led to a large number of reactive oxygen species (ROS) produced. Excessive ROS directly damage cell membranes, DNA and proteins, leading to the change and loss of cell function and cause growth inhibition and induction of apoptosisMangiferin is a four-hydroxypyridine carbon glycoside, a double benzene pyridine ketones. Modern pharmacological and clinical studies have shown that mangiferin and mangiferin containing various plant extracts with the physiological and pharmacological effects, such as lipid peroxidation, anti-cancer, anti-diabetic, anti-inflammatory anti-virus, stop-coughing, immune regulation.Using bone marrow mesenchymal stem cells for cell transplantation, cell viability is low. In hypoxia, the introduction of antioxidants on the cells exercise antioxidant, anti-apoptotic protection. Mangiferin as a promising anti-apoptotic agent, to enhance the survival of cells transplanted stem cells provide a theoretical basis and practical experience.Although the oxidation of mangiferin at home and abroad research has accumulated some data, the protected mechanism of mangiferin in bone marrow mesenchymal stem cells in in hypoxic injury has not been reported.The subject of the research base in vitro, using cell biology and modern molecular biology techniques, testing the effect hypoxia model induced by cobalt chloride in vitro environment on rat bone marrow mesenchymal stem cells biology.To investigate the protection mechanism of mangiferin on rat bone marrow mesenchymal stem cell. With mangiferin pretreatment, the protection of mangiferin on rat bone marrow mesenchymal stem cell in apoptosis and oxidative stress were observed;real-time fluorescence relative quantitative RT-PCR and Western-blot method s were used to detect the expression of gene and protein related to apoptosis and oxidative stress.Include the following four parts Part I Culture and identification of rat bone marrow mesenchymal stem cellsOBJECTIVE: To establish a method of isolation, cultivation and purification of rat BMSCs in vitro, to observe cell morphology, and to assess surface markers and multi-directional differentiation capacity.METHODS: BMSCs from rats were isolated, cultured and purified by the whole bone marrow adherence method, for morphology observations, the growth curve was drawn, cell cycle was analyzed, cell surface markers were assessed by flow cytometry, and BMSCs were induced to differentiate into osteoblasts and adipocytes.RESULTS: BMSCs from rats were spindle cell-based, showing radial colony arrangement. Cells kept strong growth and could passage in continuous and stable manner over 10 passages. The growth curve and cell cycle demonstrated that BMSCs were consistent with the growth characteristics and good activity of normal cells. At the third passage, BMSCs were negative for CD34 and CD45, but positive for CD44, CD90 and CD105. Following induction, oil red O staining, alkaline phosphatase staining, von Kossa staining and alizarin red staining produced a strong reaction in cells. Whole bone marrow adherence method is simple and can isolate, purify and amplify BMSCs in vitro. The obtained cells have general biological characteristics of mesenchymal stem cells, and also have potentiality of multi-directional differentiation.CONCLUSION:This experimental method has important practical significance to provide adequate source of seed cells for tissue engineering. Part II The extraction, identification, pharmacokinetics of Mangiferin and the biological characteristic effects of mangiferin on rat bone marrow mesenchymal stem cell.OBJECTIVE: To investigate the extraction, identification, pharmacokinetics of Mangiferin and the biological characteristic effects of mangiferin on rat bone marrow mesenchymal stem cell.METHODS: By ethanol extraction and after extraction, pretreatment, regeneration, washing and recrystallization, mangiferin was obtained. Through ultraviolet and Infrared absorption spectroscopy, nuclear magnetic resonance, high performance liquid chromatography (HPLC) , identified the chemical structure and content of the extract.By oral administration, the pharmacokinetics of mangiferin in rat blood was evaluated.By drug cytotoxicity test (MTT), cell cycle analysis, cell proliferation, the biological effects of mangiferin on rat bone marrow mesenchymal stem cells were detected.RESULTS:Extracted by refluxing with ethanol ,the concentrations of mangiferin can up to 98.0% and 94.9%, and the purity has meet the need for efficacy trials.High performance liquid chromatography is a effective and feasible method in measuring content of mangiferin in mouse plasma.The low concentrations of mangiferin (20-80μmol/l) can promote the growth of bone marrow mesenchymal stem cells ,to 40μmol / l mangiferin for the best. The high concentration of mangiferin inhibit cell growth, with a concentration-dependent.Conclusion: By using ethanol , the high purity of mangiferin was obtain, and by high performance liquid chromatography, mangiferin could be detected . Mangiferin have a certain influence on cell growth.Part III The protection mechanism of mangiferin in hypoxia induced apoptosis in rat bone marrow mesenchymal stem cellsOBJECTIVE:To investigate the protection mechanism of mangiferin in hypoxia induced apoptosis in rat bone marrow mesenchymal stem cells METHODS: Established hypoxia model of cells.The apoptosis rate by MTT,apoptosis detection of flow cytometry, detection of mitochondrial membrane potential of flow cytometry were observed.the differences of mRNA and protein expressions of Casapse3,Casapse8,Casapse9,Bcl-2 and Bax were assayed by real-time fluorescence relative quantitative RT-PCR and Western-blot method.RESULTS: Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of hypoxic injury in a dose dependent manner. With the increase of the concentration of mangiferin ,the apoptosis rate in rat bone marrow mesenchymal stem cells in hypoxic injury model were gradually reduce. (P<0.01).Cobalt chloride(CoCl2) can increase Casapse3, Casapse8, Casapse9, Bax gene and protein expression, decrease Bcl-2 gene and protein expression. Mangiferin inhibited cobalt chloride induced increasing of Casapse3, Casapse8, Casapse9, Bax gene and protein expression, and decreasing of Bcl-2 gene and protein expression(P<0.01).CONCLUSION:Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of hypoxic injury in a dose dependent manner.Part IV The antioxidant protection mechanism of mangiferin in rat bone marrow mesenchymal stem cells in hypoxiaOBJECTIVE:To investigate the antioxidant protection mechanism of mangiferin in hypoxia in rat bone marrow mesenchymal stem cellsMETHODS: Established hypoxia model of cells. By UV-Vis spectrophotometer , the relevant indicators of oxidative stress: superoxide dismutase(SOD), malondialdehyde(MDA), glutathione peroxidase(GSH-XP), catalase(CAT) were observed.in cells and cell culture medium. reactive oxygen species (ROS) were measured by flow cytometry.The differences of mRNA and protein expressions of RelA,HIF-1αand Hsp70 were assayed by real-time fluorescence relative quantitative RT-PCR and Western-blot method.RESULTS: Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of oxidative stress in a dose dependent manner.With the increase of the concentration of mangiferin ,the oxidative stress in rat bone marrow mesenchymal stem cells in hypoxic injury model were gradually reduce. ( P<0.01 ) .Cobalt chloride(CoCl2) can increase RelA,HIF-1αand Hsp70 gene and protein expression. Mangiferin inhibited CoCl2 induced increasing of RelA and Hsp70x gene and protein expression(P<0.01);but mangiferin can not inhibite HIF-1αgene and protein expression(P<0.05).CONCLUSION:Mangiferin have a protective effect on hypoxia-induced oxidative stress on rat bone marrow mesenchymal stem cells, in a dose dependent manner.
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