PTEN基因沉默对BMSC氧糖剥夺模型的保护作用及其对Bcl-2凋亡通路的影响

摘要

目的 研究RNA干扰沉默第10号染色体同源丢失性磷酸酶张力蛋白(PTEN)基因对骨髓间充质干细胞(BMSC)氧糖剥夺模型的保护作用,并探讨其对Bcl-2敏感的凋亡通路的影响,分析其保护作用的机制.方法体外分离培养BMSC并鉴定,通过脂质体法转染PTEN基因特异的siRNA沉默PTEN基因；RT-PCR检测PTEN基因的表达；制备体外氧糖剥夺模型观察PTEN基因的沉默对细胞的保护作用.Western blot检测Bcl-2蛋白表达的变化.结果成功培养BMSC并鉴定.RT-PCR检测结果显示siRNA成功干扰了PTEN基因的表达,流式细胞仪检测结果显示PTEN基因沉默后的细胞对氧糖剥夺模型有较强的耐受能力,Western blot检测结果显示Bcl-2蛋白明显增多.结论PTEN基因的沉默使BMSC更能耐受氧糖剥夺的环境,增强了细胞的存活能力,其机制可能是通过Bcl-2敏感的凋亡通路抑制细胞凋亡.%Abstract Objective To study the cell protective effect of of PTEN silencing by RNAi on BMSC oxygen and glucose deprivation model and analyze its mechanism, and to explore its influence on Bcl-2 apoptosis pathway. Methods BMSCs were isolated and identified in vitro. We transfected specific siRNA of P7EJV gene into the BMSCs via liposomes and detected the change of the mRNA of PTEN by RT-PCR. The protective effect of PTEN silencing on the oxygen and glucose deprivation model of BMSCs was observed. The change of Bcl-2 protein was detected by Western blot. Results We cultured and identificated the BMSCs successfully in vitro. RT-PCR showed that the mRNA expression of PTEN dramatically declined in the RNAi group; FCM showed that the apoptosis was decreased significantly in the RNAi cells; Western blot showed that the Bcl-2 protein expression dramatically increased in the RNAi group. Conclusion PTEN silencing exerts strong protective effect on the oxygen and glucose deprivation model of BMSCs,and enhances the survive ability by inhibiting cell apoptosis,the mechanism of which may be the inhibition of Bcl-2 apoptosis pathway.