端粒酶催化亚单位基因启动子调控Hsv—tk/GCV系统对肺癌细胞A549选择性杀伤作用的研究  

A study on selective killing effect of Hsv-tk/GCV driven by human telomerase catalytic subunit promoter on human lung cancer cell A549

作　　者：唐小军 王艳萍[2] 周清华[1] 车国卫[1] 陈小禾[3] 朱大兴[1] 

机构地区：[1]四川大学华西医院胸心外科,成都610041 [2]四川大学华西医院肿瘤分子诊断研究室,成都610041 [3]四川大学华西医院干细胞与组织工程研究室,成都610041

出　　处：《中华医学遗传学杂志》2007年第2期148-152,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（30270589,30470762）

摘　　要：目的研究人端粒酶催化亚单位（human telomerase catalytic subunit，hTERT）基因启动子调控单纯疱疹胸苷激酶／更昔洛韦（herpes simplex virus-thymidine kinase／ganciclovir，Hsv-tk／GCV）治疗系统对人肺癌细胞株A549的选择体外杀伤作用。方法（1）用脂质体法将hTERT启动子和sv40启动子调控的比基因表达质粒（pGL3-hTp-tk和pGL3-sv40-tk）转染端粒酶阳性的人肺腺癌细胞株A549及端粒酶阴性的人胚肺成纤维细胞株MRC-5，用逆转录-PCR方法检测转染细胞中此基因的表达情况；（2）用MTT法检测GCV对上述转染细胞体外增殖的抑制作用；（3）用流式细胞仪检测GCV对上述转染细胞凋亡和细胞周期的影响。结果（1）转染pGCS-sv40-tk的细胞A549、MRG5和转染pGCS-hTp-tk的A549均有tk mRNA表达，转染pGL3-hTp-tk的MRC-5无tk mRNA表达；（2）GCV对转染pGL3-sv40-tk的细胞A549、MRC-5和转染pGCS—hTp-tk的A549体外增殖均有明显抑制作用，对转染pGL3-hTp-tk的MRG-5无明显抑制作用；（3）转染pGL3-sv40-tk的A549、MRC-5和转染pGL3-hTp-tk的A549细胞，GCV处理后细胞凋亡指数（21．58％、9．35％和23．19％）均显著高于转染pGL3-hTp的A549和MRC-5细胞（0．78％和0．55％）及空白对照A549和MRC-5细胞（2．17％和0．60％），转染pGL3-hTp-tk的MRC-5细胞凋亡指数（0．88％）无明显升高。结论hTERT启动子调控Hsv-tk基因可以在肺癌细胞中选择性表达，hTERT调控的Hsv-tk／GCV治疗系统对肺癌细胞体外增殖具有靶向性抑制作用。Objective To study selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir （Hsv-tk/GCV） driven by human telomerase catalytic subunit （hTERT） promoter on lung cancer cell line A549 in vitro. Methods （1） Expression plasmids of Hsv-tk gene driven by hTERT promoter and sv40 promoter respectively（pGL3- hTp-tk and pGL3-sv40-tk）were transfected into telomerase-positive human lung adenocarcinoma cell line A549 and telomerase-negative fetal lung fibroblast cell line MRC-5. Reverse transcription-PCR was performed to detect expression of tk gene in above transfected cell hnes; （2） Inhibition effect on proliferation of above transfected cell lines treated with GCV was investigated with MTT method; （3） Influence of GCV on apeptosis and cell cycle of above transfected cell lines was investigated with flow cytometry. Results （1） tk mRNA expression was detected in both A549 and MRC-5 transfected with pGL3-sv40-tk, also in A549 transfected with pGL3-hTp-tk, but not in pGL3-hTp-tk transfected MRC-5; （2） GCV showed significant inhibition effects on proliferation of pGL3-sv40-tk transfected A549 and MRC-5 in vitro, also on that of pGL3-hTp-tk transfected A549, but not on that of pGL3-hTp-tk transfected MRC-5; （3） Treated with GCV, apeptosis index （AI） of pGL3-sv40-tk transfected A549 and MRC-5 as well as pGL3-hTp-tk transfected A549 （21.58%, 9.35% and 23.19% respectively） increased significantly, compared with A549, MRC-5 transfected with pGL3-hTp （0.78%and 0.55 % respectively） and A549, MRC-5 without plasmid transfection as blank control （2.17 % and 0.60% respectively） ; GCV had no influence on AI of pGL3-hTp-tk transfected MRC-5（0.88%）. Conclusion tk gene driven by hTERT promoter could express selectively in lung cancer cell A549. Hsv-tk/GCV driven by hTERT promoter could selectively inhibit proliferation of lung cancer cell.

关 键 词：肺癌 自杀基因 端粒酶催化亚单位 选择性表达 

分 类 号：R686[医药卫生—骨科学]