The Research Of Ethanol Induced Rats Bone Marrow Stem Cell Appoptsis Mechanism

OBJECTIVE: This study is to establish to separate in vitro and purificate,culture expansion,induce and differentiate rat's BMSCs by whole bone marrow adherent culture ,and to identify the cells to established the cellular basis for further research.METHODS: Applications the method of separation of whole bone marrow culture adherent cells to separate and culture the SD rat's bone marrow mesenchymal stem cell.Identificate the BMSCs cell surface antigen:CD44,CD90,CD105,CD34,CD45.The BMSCs cell of Adipogenic induction was identificated by oil red O staining.The BMSCs cell of Osteogenic identificated by Alkaline phosphatase staining and Stained with silver nitrate.METHODS: High purity of SD rat's bone marrow mesenchymal stem cells can be isolated and culture by the method of separation of whole bone marrow culture.The BMSCs cell's activity are more than 95% after the separation cell.FCM was used to detect the CD44 ,CD90, CD105-positive cells of rat bone marrow mesenchymal stem cells were 99.34%, 99.34%, 99.35%;CD34, CD45 negative expression were 1.47%, 1.56%;After the 9th day of BMSCc adipogenic, The cell morphology and induction of oil red O staining cells were seen to form a large number of lipid droplets.After the 12th day of BMSCc adipogenic,cell mass accumulation and a dense opaque shadow were visibled by cell morphology ,Alkaline phosphatase staining and stained with silver nitrate were positive expression .RESULTS AND CONCLUSION: This experiment used all of bone marrow cells adherent culture was an ideal method to culture bone marrow stem cell.The BMSCs have the character of high purity,strong vitality , biological characters stable, it has the potency of in vitro self-renewal and proliferation and multi-directiona differentiation after induction. BACKGROUND:osteogenic differentiation abilities of MSCs which decreased and adipogenic abilities of MSCs which increased may play important role in alcohol-induced osteonecrosis of the femoral head. Recent studies have shown that ethanol induced opoptosis in bone marrow-derived mesenchymal stem cells(MSCs),and decreased the number of osteoblastic cells and broken bone cells, or damaged dynamic balance between them. It may be one of the important reasons which lead to osteoporosis even Osteonecrosis of the femoral head. However, the effect and mechanism of ethanol on apoptosis in BMSCs remains unclear.OBJECTIVE: To investigate the effect of ethanol on apoptosis in rat bone marrow-derived mesenchymal stem cells( MSCs )and their mitochondrial function and to evaluate the pathway associated with the regulation of Bcl-2and Caspase-3, the determination of Superoxide dismutase and malondialdehyde ,to in order to understand the mechanism of its apoptosis.METHODS: Bone marrow stromal cells (BMSCs) isolated from Sprague-Dawley rats were treated with ethanol at doses of 0,100,200,300,400,500,600,700,800,900 mmol/L for 24 hours . cytotoxic drug experiment was performed with MTT assay. BMSCs were treated with ethanol at doses of 0, 100, 200, 300, 400, 500 mmol/L for 6, 12, 24 hours, AnnexinV/PI flow cytometry of double label method was performed to detect the apoptosis and mitochondrial membrane potential(△ψm), BMSCs were treated with ethanol at doses of 0, 427 mmol/L for 24 hours, the levels of Bcl-2 and Caspase-3 mRNA expression were determined by RT-PCR method. BMSCs were treated with ethanol at doses of 0, 427 mmol/L for 24 hours, the levels of superoxide dismutase and trace of mda expression were determined.RESULTS AND CONCLUSION: MTT assay results showed that 50% concentration of inhibition (IC50 ) of Bone marrow stromal cells (BMSCs) of Sprague-Dawley rats growed in ethanol was 427 mmol/L. The results of Annexin V/PI assay indicated that the apoptosis rates of BMSCs were higher than that of untreated group when time and dose of ethanol was increased(P < 0.05),and Mitochondrial membrane potential(△ψm)was decreased (P < 0.05).At 24 hours, the ration of Bcl-2mRNA expression was decreased.By treatment at 427 mmol/L , the decrease was dramatically compared to control group , but the caspase-3 mRNA expression was increased significantly by treatment at 427 mmol/L (P < 0.05). Compared with 0 mmol/L group, the level of superoxide dismutase was decreased in 427 mmol/L group after 24 hours, but the trace of mda expression was increased significantly by treatment at 427 mmol/L (P < 0.05). These results suggest that ethanol can induce apoptosis in BMSCs of SD rat, and the occurrence of apoptosis may be related to the mitochondrial membrane potential damage, mitochondrial dysfunction, bcl-2 and Caspase-3 activation, the damage of balance between superoxide dismutase and trace of mda.