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

发布时间：2018-05-16 06:09

本文选题：骨髓间充质干细胞 + 基因沉默　；参考：《中国病理生理杂志》2013年08期

【摘要】：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(P0.05),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(P0.05)。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)%(P0.05)。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。
[Abstract]:Aim: to investigate the effects of silencing caspase-3 on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). Methods: the recombinant shRNA lentivirus targeting caspase-3 was constructed and transfected into MSCs. The transfection results were identified by real-time PCR and Western blotting at the level of mRNA and protein. MTS assay was used to detect cell proliferation and flow cytometry to detect cell cycle. Real-time PCR was used to detect the expression of bcl-2 and bax mRNA. Hoechst fluorescence staining was used to detect apoptosis. Results the results of PCR and Western blotting showed that the stable rat MSCs cells transfected with shRNA-caspase-3 were successfully established. Silencing caspase-3 significantly increased the proliferation rate of MSCs and the percentage of S phase cells, which was 52.66 卤0.30%. After silencing caspase-3, the expression of bcl-2 mRNA up-regulated and down-regulated the ratio of bcl-2 / Bax to increase P0.05. The rate of apoptosis in the transfected group was 15.01 卤1.73, which was lower than that in the empty vector group (25.67 卤3.05) and the blank control group (23.67 卤1.16). Conclusion: silencing caspase-3 can regulate the cell cycle of MSCs, promote cell proliferation and reduce apoptosis.
【作者单位】：中山大学孙逸仙纪念医院心胸外科;广州市第一人民医院脑内科;
【基金】：广东省科技计划项目(No.2012B031800258) 中山大学青年教师培育项目(No.11ykpy30)
【分类号】：R363

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