人端粒酶逆转录酶基因沉默对卵巢癌细胞株A2780凋亡的影响及其机制  

Effect of Human Telomerase Reverse Transcriptase Gene Silence on Apoptosis of Ovarian Cancer A2780 Cells

作　　者：罗祎[1] 姚珍薇[1] 杨雅莹[2] 易永芬[2] 

机构地区：[1]重庆医科大学附属第一医院妇产科,重庆400016 [2]重庆医科大学病理教研室分子医学与肿瘤研究中心,重庆400016

出　　处：《中国生物制品学杂志》2011年第7期749-756,共8页Chinese Journal of Biologicals

基　　金：教育部博士点基金(20060631006)

摘　　要：目的检测人端粒酶逆转录酶(Human telomerase reverse transcriptase,hTERT)基因沉默对卵巢癌细胞株A2780生长抑制和凋亡的影响,并探讨其与p53、p21基因表达激活的相关性。方法设计合成针对hTERT基因的shRNA干扰质粒,与可表达荧光蛋白的pGenesil-1.1质粒连接,构建重组质粒pG1、pG2和阴性质粒pGCR,转染A2780细胞,采用荧光定量RT-PCR检测转染细胞hTERT基因mRNA的表达,Western blot检测细胞hTERT、P53和P21蛋白的表达,TRAP法检测细胞的端粒酶活性,CCK-8法检测细胞的生长,流式细胞术分析细胞周期,AnnexinⅤ-PE/7AAD双染和TUNEL法检测细胞凋亡。结果酶切及测序结果证实重组质粒pG1、pG2和pGCR构建正确,质粒pG1和pG2转染可明显下调A2780细胞hTERT的表达水平和端粒酶活性,而上调细胞P53和P21蛋白的表达水平;沉默hTERT基因表达能够引起A2780细胞的生长抑制和G0-G1期细胞比例明显下降,并出现明显的细胞凋亡。结论针对hTERT基因的shRNA干扰质粒在体外能有效和特异地沉默卵巢癌A2780细胞hTERT基因的表达,降低端粒酶活性,并引起细胞的生长抑制和凋亡,其机制可能与抑癌基因p53及p21上调有关。Objective To investigate the effect of human telomerase reverse transcriptase（hTERT） gene silence on growth and apoptosis of ovarian cancer A2780 cells as well as the relationship between the effect and activation of p53 and p21 gene expressions.Methods The shRNA interfering plasmid specific to hTERT gene was designed and synthesized,then linked to plasmid pGenesil-1.1 expressing fluorescent protein.A2780 cells were transfected with the constructed recombinant plasmids pG1,pG2 and negative control plasmid pGCR respectively,then determined for expression of hTERT mRNA by RT-PCR,for expressions of hTERT,P53 and P21 proteins by Western blot,for telomerase activity by TRAP method,for growth by CCK-8 method,for cell cycle by flow cytometry,and for apoptosis by Annexin Ⅴ-PE/7AAD staining and TUNEL method.Results Restriction analysis and sequencing proved that recombinant plasmids pG1 and pG2 were constructed correctly.The transfection with pG1 and pG2 decreased the hTERT expression level and telomerase activity,while increased the expression levels of P53 and P21 proteins.The silence of hTERT gene expression inhibited the growth,decreased the percentage at G0-G1 stages,and induced significant apoptosis of A2780 cells.Conclusion The shRNA interference plasmid specific to hTERT gene silenced the hTERT gene expression in A2780 cells effectively and specifically,decreased the activity of telomerase,and caused the growth inhibition and apoptosis of A2780 cells,by a potential mechanism associated with up-regulating the expression of cancer suppressor genes p53 and p21.

关 键 词：人端粒酶逆转录酶 基因沉默 卵巢肿瘤 细胞增殖 细胞凋亡 

分 类 号：R737.31[医药卫生—肿瘤]