慢病毒介导的caspase-3沉默对大鼠骨髓间充质干细胞增殖和凋亡的影响

 目的：探讨沉默caspase-3对大鼠骨髓间充质干细胞（MSCs）增殖、细胞周期和凋亡的影响。方法：构建靶向caspase-3的shRNA重组慢病毒并转染MSCs，通过real-time PCR和Western blotting 在mRNA及蛋白水平鉴定转染结果。采用MTS法检测细胞增殖，流式细胞术检测细胞周期。Real-time PCR检测bcl-2和bax mRNA的表达。Hoechst荧光染色法检测细胞的凋亡情况。结果：Real-time PCR和Western blotting结果均表明成功建立稳定转染shRNA-caspase-3的大鼠MSCs细胞株。沉默caspase-3使MSCs的增殖率明显提高（P＜0.05），且S期细胞百分比明显增多，为（52.66±0.30）%。沉默caspase-3后bcl-2 mRNA表达上调，bax mRNA表达下调，bcl-2/bax比值升高（P＜0.05）。转染组的细胞凋亡率为（15.01±1.73）%，低于空载体组的（25.67±3.05）%和空白对照组的（23.67±1.16）%（P＜0.05）。结论: 沉默caspase-3能调控MSCs的细胞周期，促进细胞增殖，减少细胞凋亡。

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.