Yu Lili, Wang Zehua
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2009 Oct;29(5):625-30. doi: 10.1007/s11596-009-0518-1. Epub 2009 Oct 11.
The recombinant plasmids pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into HeLa cells and cisplatin was added with different concentrations in order to study the inhibitory effects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression of Bcl-2, Bax, caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into HeLa cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations (3.0, 6.0 and 9.9 microg/mL) was added into the transfected HeLa cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-1-BIRC71 and pGenesil-1-BIRC72 into HeLa cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was significantly increased in transfection group as compared with control group (P<0.05). Cisplatin could increase the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expression levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were increased in transfection group as compared with those in control group (P<0.05). It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in HeLa cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2 and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the process of apoptosis induction.
将重组质粒pGenesil-1-BIRC71和pGenesil-1-BIRC72转染入HeLa细胞，并加入不同浓度的顺铂，以研究Livin基因的抑制作用，增加顺铂诱导的细胞凋亡，并检测Bcl-2、Bax、caspase-3和survivin基因的表达。将pGenesil-1-BIRC71和pGenesil-1-BIRC72转染入HeLa细胞，采用荧光定量实时PCR检测Livin、Bcl-2、Bax、caspase-3和survivin基因的表达水平。然后将不同浓度（3.0、6.0和9.9μg/mL）的顺铂加入转染后的HeLa细胞中，24小时和48小时后，通过流式细胞术检测细胞凋亡率。将pGenesil-1-BIRC71和pGenesil-1-BIRC72转染入HeLa细胞后，Livin基因的表达水平明显降低，转染组的细胞凋亡率与对照组相比显著升高（P<0.05）。顺铂可呈剂量和时间依赖性地增加细胞凋亡率。加入顺铂后，转染组中Bcl-2 mRNA的表达水平降低，而Bax、caspase-3和survivin mRNA的表达水平与对照组相比升高（P<0.05）。结论是，靶向Livin基因的shRNA表达载体可抑制HeLa细胞中Livin基因的表达，并增强顺铂诱导的细胞凋亡，这与Bcl-2表达降低以及Bax和caspase-3的激活有关。Survivin可能作为凋亡诱导过程中的拮抗剂发挥重要作用。
