重组人Bcl2及Ercc1基因腺病毒的构建及对肺癌细胞凋亡的影响  

Construction of siBcl2-Ercc1 recombinant adenovirus and its effects on apoptosis of lung cancer cells

作　　者：王芳[1] 刘瑾[1] 

机构地区：[1]山西医科大学公共卫生学院,太原市030001

出　　处：《实用医学杂志》2015年第15期2443-2446,共4页The Journal of Practical Medicine

基　　金：山西省青年科学基金项目(编号:2012021012-3)

摘　　要：目的:通过RNA干扰技术沉默B淋巴细胞瘤-2基因(Bcl2)和切除修复交叉互补基因1(Ercc1)并观察对肺癌细胞株增殖的影响。方法 :分别设计特异性沉默Bcl2和Ercc1的短发夹RNA序列,并克隆到腺病毒骨架载体上形成重组腺病毒质粒。将质粒转染到HEK293细胞中,包装和扩增形成完整高滴度的重组腺病毒,将腺病毒感染人肺癌H460细胞株,流式细胞术检测抑制Ercc1和Bcl2基因后肺癌细胞凋亡变化。结果:通过酶切鉴定,证实重组腺病毒载体构建成功,PCR鉴定扩增出1 200 bp的目的条带,病毒滴度为5×1011/m L。流式细胞术检测结果显示,Adsi Bcl2-Ercc1组细胞凋亡率比单基因干扰组增高。结论:成功构建干扰Bcl2和Ercc1的重组质粒及重组腺病毒,同时沉默Bcl2和Ercc1可以促进肺癌细胞凋亡。Objective Toexplore the effect of Bcl2 and Erccl gene on apoptosis by RNA interference of Bcl2 and Erccl gene expression in non-small cell lung cancer cell line. Methods shRNA targeting Bel2 and Erecl gene were cloned into vector to construct recombinant adenovirus plasmids which were transfected into HEK293 cells. High titer adenovirus was obtained after package and amplification. H460 cells were infected with the adenovirus and apoptosis rate was detected by flow cytometry. Results The results of restriction endonuclease digestion showed that recombinantadenovirus vector adsiBcl2-Erccl was successfully constructed. The positive 1 200 bp amplification bands could be seen in PCR analysis, and the titer of the adenovirus was 5 ×10^11/mL. The apoptosis rate of AdsiBcl2-Erccl was higher than that of the two single silenced gene groups. Conclusions The recombinant plasmids and recombinant adenovirus were successfully constructed. Silencing of Bcl2 and Erccl through shRNA promotes the apoptosisin lung cancer cells.

关 键 词：肺肿瘤 凋亡 重组腺病毒 H460细胞 

分 类 号：R734.2[医药卫生—肿瘤]