Bcl-2基因沉默对A549细胞化疗药物杀伤敏感性的影响  

The effect of Bcl-2 gene silencing on the sensitivity of cell line A549 to chemotherapeutic drugs

作　　者：王姣琦[1] 杜珍武[1] 高娴绯[1] 吴玫[1] 张玉成[1] 潘颖 王茜 张桂珍[1] 

机构地区：[1]吉林大学中日联谊医院中心实验室,长春130033 [2]妇产科 [3]肾内科

出　　处：《中华结核和呼吸杂志》2013年第3期191-197,共7页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：吉林省卫生厅科研课题（20092081）

摘　　要：目的探讨抑制Bcl-2基因表达对人肺癌A549细胞化疗药物杀伤敏感性及其相关基因mRNA表达的影响。为进一步提高非小细胞肺癌（NSCLC）化疗疗效提供实验依据。方法构建靶向人Bcl-2基因的微小RNA重组质粒，并转染至A549细胞，设稳定转染的实验组、阴性对照质粒组和空白对照组，运用逆转录聚合酶链反应（RT-PCR）琼脂糖凝胶电泳、实时荧光定量PCR和蛋白质免疫印迹法（Westernblot）检测转染细胞中Bcl-2mRNA转录和蛋白的表达水平，四甲基偶氮唑蓝（MTT）法和流式细胞术分析转染后细胞的增殖和周期变化。MTT法检测干扰后A549细胞对依托泊苷、5-氟尿嘧啶、顺铂、阿霉素、长春新碱、紫杉醇及长春瑞滨等7种化疗药物敏感性的变化。应用RT-PCR及实时荧光定量PCR方法进行核苷酸切除修复交叉互补基因1（ERCC1）、胸腺嘧啶合成酶（TYMS）、Ⅲ型β-微管蛋白（Class Ⅲ β-tubulin）、拓扑异构酶2α（TOP2α）等化疗敏感性相关基因mRNA表达的检测。结果成功构建微小RNA表达载体，稳定转染A549细胞后，实验组Bcl-2mRNA表达（相对表达量为0．002±0．001）与阴性对照质粒组（相对表达量为0．104±0．003）相比下降98．1％，蛋白表达水平下调57．6％（t值分别为98．70和7．66，均P〈0．05）。流式细胞术测定实验组细胞周期阻滞在G1期，MTT法检测示实验组细胞生长受到明显抑制，对依托泊苷、顺铂、紫杉醇、长春瑞滨等4种药物的敏感性明显增加[实验组IC50分别为（107．3±0．1）mg／L、（7．7±0．6）mg／L、（11．5±1．9）mg／L和（10．8±1．6）mg／L，阴性对照质粒组IC50分别为（145．8±0．1）mg／L、（60．7±1．4）mg／L、（80．6±1．7）mg／L和（20．6±1．7）mg／L]，差异均有统计学意义（t值分别为655．33，108．04，82．16和12．48，均P〈0．05）。此外，Bcl-2表达下调还使实验组ERCCl、TYMS、TOObjective To investigate the effects of miRNA-mediated down-regulation of the Bcl-2 gene on the chemotherapeutic sensitivities and mRNA transcriptions of sensitivity associated genes in human lung adenocarcinoma cell line A549 ceils, and therefore to provide experimental data for improving the chemotherapeutic effects on non-small cell lung cancer （NSCLC）. Methods The miRNA recombinant plasmid targeting to human Bcl-2 gene was designed, synthesized and stably transferred into A549 cells by lipofectin technique as the experiment group. The transcription of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）by agarose gel electrophoresis, real-time PCR, and the protein level of Bcl-2 was measured by Western blot to confirm the function of miRNA plasmid. The cell proliferation was examined by methyl thiazolyl tetrazolium （MTF） assay. Cell cycle was measured by flowcytometry. Drug sensitivities of A549 cells to etoposide, 5-fluorouracil, cisplatin, adriamycin, vincristine, paclitaxel and navelbine were analyzed by MTF assay. The mRNA expressions of excision repair cross- complementing gene 1 （ERCC1）, thymidylate synthase（TYMS）, Class Ⅲ β-tubulin, topoisomerase 2 alpha （TOP2α） genes were detected by RT-PCR and real-time PCR. Results The recombinant miRNA plasmid was successfully synthesized and stably transferred into A549 cells. The transcription of Bcl-2 mRNA dramatically decreased by 98.1% in the experiment group（ RQ = 0. 002 ± 0. 001 ） compared to that in the negative control group（ RQ = 0. 104 ± 0. 003 ） by real-time PCR （ t = 98.70, P 〈 0.05 ） ; and the protein level of Bcl-2 in the experiment group decreased by 57.6% by Western blot（t =7.66,P 〈0. 05）. The cell cycle profile showed that the low expression of Bcl-2 gene led to A549 cell cycle arrest at Gl-phase. The results of MTT showed that the growth of A549 cells in the experiment group was markedly inhibited. The sensitivities of A549 cells to etoposide, cisplatin, paclitaxel,

关 键 词：基因 BCL-2 抗肿瘤药 RNA干扰 基因 

分 类 号：R734.2[医药卫生—肿瘤]