hTERT启动子特异性调控炭疽毒素致死因子表达以杀伤人肺腺癌A549细胞的研究  

Specific regulation of anthrax toxin lethal factor expression by hTERT promotor in human lung carcinoma A549 cells

作　　者：卓文磊[1] 陈芳琳[1] 陶光利[2] 

机构地区：[1]第三军医大学新桥医院全军肿瘤研究所,重庆400037 [2]第三军医大学新桥医院全军肾脏病中心,重庆400037

出　　处：《重庆医学》2012年第11期1051-1054,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(30800506);重庆自然科学基金资助项目(CSTC;2008BB5124);第三军医大学科研创新基金资助项目(2008D204)

摘　　要：目的观察人端粒酶逆转录酶启动子(hTERTp)靶向调控炭疽毒素致死因子(LF)表达对人肺腺癌A549细胞活力及凋亡/坏死的影响。方法构建含hTERTp或CMV启动子(CMVp)且表达LF的真核质粒表达载体(phTERTp-LF或pCMV-LF),分别转染人肺腺癌A549细胞株和正常人胚肺成纤维MRC-5细胞株。针对A549、A549hTERTp-LF、A549CMVp-LF、MRC-5、MRC-5hTERTp-LF、MRC-5CMVp-LF六组细胞,分别采用RT-PCR和Western blot检测LF mRNA和蛋白表达,用MTT分析检测细胞活力,用流式细胞仪检测细胞凋亡/坏死。结果与A549组比较,A549hTERTp-LF组、A549CMVp-LF组均出现明显的LF mRNA和蛋白表达,以及细胞活力降低和凋亡/坏死率上升;与A549hTERTp-LF组相比,A549CMVp-LF组LF mRNA和蛋白表达更高,细胞活力降低和凋亡/坏死率上升更明显。与MRC-5组比较,MRC-5hTERTp-LF组也不能测出LF mRNA和蛋白表达,细胞活力和凋亡/坏死率亦无明显变化,但MRC-5CMVp-LF组的LF mRNA和蛋白表达上升,细胞活力降低和凋亡/坏死率增加。结论 hTERTp能特异性地驱动LF表达并杀伤人肺腺癌A549细胞,但避免了对MRC-5细胞的毒性,可望提供一种针对肺腺癌细胞且不良反应小的靶向治疗策略。Objective To investigate the effects of anthrax toxin lethal factor（LF） expression regulated by human telomerase reverse transcriptase gene promoter（hTERTp） on A549 cells viability and apoptosis/necrosis.Methods LF eukaryotic expression vectors for LF,phTERTp-LF containing hTERTp or pCMV-LF containing CMV promoter（CMVp）,was designed,constructed,and lipotransfected into human lung adenocarcinoma cells line（A549 cells） or human lung embryonated fibroblast cells line（MRC-5 cells） respectively.For the six groups of cells,including A549,A549hTERTp-LF,A549CMVp-LF,and MRC-5,MRC-5hTERTp-LF,MRC-5CMVp-LF.LF mRNA and protein expression were examined by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot,cell viability was measured by MTT analysis,apoptosis/necrosis was detected using Annexin V-FITC labeling method.Results Compared with that in A549 group,LF mRNA and protein expression upregulated,cell viability decreased,and apoptosis/necrosis rates increased significantly in A549hTERTp-LF and A549CMVp-LF groups.Compared with that in A549hTERTp-LF group,there are stronger LF mRNA and protein expression,lower cell viability,and higher apoptosis/necrosis rate in A549CMVp-LF group.Compared with that in MRC-5 group,LF mRNA and protein expression were not detectable also,cell viability and apoptosis/necrosis rates are similar in MRC-5hTERTp-LF group,however,LF mRNA and protein expression upregulated,cell viability decreased,and apoptosis/necrosis rates increased significantly in MRC-5CMVp-LF group.Conclusion Although activity is weaker than CMVp,hTERTp can specifically drive LF expression,injure human lung adenocarcinoma A549 cells,and avoid the toxicity to MRC-5 cells.This study may provide a targeted strategy for lung adenocarcinoma therapy with low side effect.

关 键 词：人端粒酶逆转录酶 启动区 致死因子 细胞凋亡 A549细胞 

分 类 号：R734.2[医药卫生—肿瘤]