乙醇诱导大鼠骨髓间充质干细胞的凋亡机制

Apoptotic mechanism of rat bone marrow mesenchymal stem cells induced by ethanol

背景:有研究表明乙醇可诱导骨髓间充质干细胞凋亡并引起成骨细胞和破骨细胞数量减少,但乙醇对骨髓间充质干细胞凋亡的影响以及作用机制目前尚不十分清楚。目的:观察乙醇对大鼠骨髓间充质干细胞凋亡、线粒体功能的影响及bcl-2、Caspase-3表达的变化。方法:应用全骨髓培养法分离培养SD大鼠骨髓间质干细胞,置于0,100,200,300,400,500,600,700,800,900mmol/L乙醇中作用24h,MTT法进行细胞毒性药物实验;置于0,100,200,300,400,500mmol/L乙醇中作用6,12,24h,AnnexinV/PI双标记法流式细胞仪检测细胞凋亡和线粒体膜电位变化情况;置于0,427mmol/L乙醇中作用24h,RT-PCR法检测与凋亡有关的基因bcl-2和Caspase-3mRNA表达水平。结果与结论:MTT检测结果显示427mmol/L是乙醇对大鼠骨髓间充质干细胞生长的半数抑制浓度;AnnexinV/PI检测结果表明,与0mmol/L组比较,随作用时间的延长与乙醇浓度的增加,骨髓间充质干细胞凋亡率及线粒体跨膜电位的破坏水平明显升高(P<0.05)。与0mmol/L组比较,乙醇作用24h后427mmol/L组bcl-2mRNA表达水平下降,Caspase-3mRNA表达水平增加(P<0.05)。说明乙醇能诱导大鼠骨髓间充质干细胞凋亡,凋亡的发生可能与线粒体膜电位破坏、线粒体功能障碍、bcl-2和Caspase-3激活有关。

BACKGROUND：Previous studies demonstrated that ethanol can induce apoptosis in bone marrow mesenchymal stem cells（BMSCs）,and decrease the number of osteoplasts and osteoclasts.However,the effect and mechanism of ethanol on apoptosis in BMSCs remains unclear.OBJECTIVE：To investigate the effect of ethanol on apoptosis in BMSCs of rats and their mitochondrial function and to evaluate the pathway associated with the regulation of Bcl-2 and Caspase-3.METHODS：BMSCs were 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,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.RESULTS AND CONCLUSION：MTT assay results showed that 50% concentration of inhibition（IC50） of BMSCs of rats grew in ethanol was 427 mmol/L.The results of Annexin V/PI assay indicated that the apoptosis rates of BMSCs and mitochondrial membrane potential were higher than that of untreated（0 mmol/L） group when time and dose of ethanol was increased（P 0.05）.Compared with 0 mmol/L group,the level of Bcl-2mRNA expression was decreased in 427 mmol/L group after 24 hours,but the caspase-3 mRNA 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.