siRNA对肺癌细胞株bcl-2基因表达的抑制  

siRNA Inhibited bcl-2 Expression in Human Lung Cancer Cell Lines

作　　者：胡海燕[1] 张洹[1] 

机构地区：[1]暨南大学医学院血液病研究所,广州510623

出　　处：《上海第二医科大学学报》2005年第5期483-486,共4页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：广东省自然科学重点基金(021195)资助项目.

摘　　要：目的研究小干扰RNA(siRNA)对bcl-2基因表达的抑制作用。方法利用Ambion公司提供的设计软件和试剂盒,设计合成以bcl-2基因为靶标的siRNA,通过脂质体将合成的siRNA转入A549和NCIH460细胞株,以未转染细胞和转染bcl2的反义药物G3139为对照,经MTT法检测siRNA对细胞生长的抑制,RTPCR检测bcl2mRNA水平,流式细胞仪检测细胞周期的改变和bcl-2蛋白表达,反应siRNA对bcl-2表达的抑制效应。结果siRNA组与对照组细胞存活率各时间点均有显著差异(P<0.05);与反义组在24、48h也有显著差异(P<0.05),而72h无差异(P>0.05)。转染12h后,siRNA组bcl-2mRNA与对照组和反义组有显著差异(P<0.05)。转染48h后,siRNA组bcl2蛋白阳性率明显低于对照组和反义组,siRNA组和反义组细胞阻滞于S期。结论体外转录合成的siRNA可抑制A549和NCIH46细胞bcl2基因的表达,效率可达50%以上。Objective To evaluate the effectiveness of small interference RNA (siRNA) on inhibiting bcl-2 gene expression. Methods With Ambion’s software and kit, we designed and synthesized siRNA targeted bcl-2, which were transfected into human lung cell lines A549 and NCI-H460 with Lipofectamine. The non-transfected cells and treatment with antisense drug G-3139 were taken as controls. MTT was used to detect the inhibitive rate of cells growth. The flowcytometric method was used to detect the change of cell cycling. The inhibitive effect of siRNA on the mRNA level by PT-PCR and on the protein level was detected by the flowcytomytric method. Results For the cell survived rate, siRNA groups were significantly lower than the control group at 24, 48, and 96 h, but only had statistic difference with the antisense group at 24, 48 h(P<0.05). As for PCR, the bcl-2 band of siRNA groups was faintes than other groups. The results of flowcytomytric method showed the positive rate of siRNA groups were significantly lower than other groups. The cells transfacted with siRNA were blocked at the S stage. Conclusion siRNA inhibited bcl-2 gene expression in NCI-H460 and A549 more than 50%.

关 键 词：BCL-2基因表达 SIRNA 肺癌细胞株 bcl-2蛋白表达 BCL-2MRNA RT-PCR检测 流式细胞仪检测 bcl-2表达 MTT法检测 mRNA水平 A549 G3139 细胞存活率 对照组 抑制作用 设计合成 反义药物 转染细胞 细胞生长 细胞周期 抑制效应 

分 类 号：R734.2[医药卫生—肿瘤]