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

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.

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

Objective:To study the cell killing effects of gene-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 MTT 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.