硝基还原酶/CB1954自杀基因系统对宫颈癌Hela细胞杀伤效应的实验研究  

The killing effects of nitroreductase/CB1954 suicide gene system on Hela cells

作　　者：滕隔玲[1] 杨业鹏[1] 李载权[1] 周梅[1] 李五岭[1] 

机构地区：[1]北京大学基础医学院放射医学教研室,北京100191

出　　处：《现代肿瘤医学》2009年第8期1403-1406,共4页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(编号:30470523)

摘　　要：目的:体外观察大肠杆菌硝基还原酶/[5-(1-氮丙啶)-2,4-二硝基苯甲酰胺](以下简称NTR/CB1954)自杀基因系统对宫颈癌Hela细胞的杀伤效应,探索一种新的宫颈癌基因治疗方法。方法:利用PCR技术从Escherichia coli K12的基因组中扩增出编码NTR的基因nfsB,酶切后,连接到真核表达载体pcDNA3上,获得重组载体pcDNA3-nfsB,lipofectamineTM2000脂质体转染法将pcDNA3-nfsB转染Hela细胞,筛选稳定表达细胞株,应用RT-PCR以及SDS-PAGE检测NTR在Hela细胞中的表达,MTT法检测NTR/CB1954对Hela细胞活力的影响,流式细胞术检测亚二倍体细胞率改变,PI/Hoechest33258双染荧光显微镜下观察Hela细胞凋亡率。结果:成功构建了真核表达载体pcDNA3-nfsB,获得稳定表达NTR的Hela细胞株,在mRNA水平以及蛋白水平检测到NTR在Hela细胞中的表达,NTR/CB1954自杀基因系统明显影响Hela细胞的活力,增加了Hela细胞的凋亡率。结论:NTR/CB1954自杀基因系统对Hela细胞在体外通过凋亡产生明显的杀伤效应。Objective：To study the cell killing effects of gcne -directed enzyme nitroreductase （NTR）/prodrug CB1954 system（GDEPS） on Hela cells,and explore a new treatment for cervical carcinoma. Methods： Escherichia coli nitroreductase gene was amplied from Escherichia k12 genome by PCR, and inserted into eukaryotic expression vector pcDNA3 to get recombinant pcDNA3 - nfsB. The recombinant was transfected into cells, and stable transfectant was gotten. NTR expression was detected by RT - PCR and SDS - PAGE in level of mRNA and protein respectively. The proliferation effect of cell and hypodiploid rate was observed respectively by M3T and flow cytometry. Apoptosis rate of cell was tested by Hoechest/PI fluorescent vital staining. Results： Nitroreductase eukaryotic expression vector pcDNA3 - nfsB was successfully constructed. Stable transfectant was selected correctly. The growth of cells was obviously effected and the apoptosis rates were significantly increased by NTR/CB1954 suicide gene system. Conclusion： The gene -directed NTR/CB1954 system might be an effective therapeutic methed for cervical carcinoma.

关 键 词：nfsB基因 硝基还原酶 CB1954 自杀基因 凋亡 

分 类 号：R730.5[医药卫生—肿瘤]