CAJ | 학술논문

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.
목적:체외관찰대장간균초기환원매/[5-(1-담병정)-2,4-이초기분갑선알](이하간칭NTR/CB1954)자살기인계통대궁경암Hela세포적살상효응,탐색일충신적궁경암기인치료방법.방법: 이용PCR기술종Escherichia coli K12적기인조중확증출편마NTR적기인nfsB,매절후,련접도진핵표체재체pcDNA3상,획득중조재체pcDNA3-nfsB,lipofectamineTM2000지질체전염법장pcDNA3-nfsB전염Hela세포,사선은정표체세포주,응용RT-PCR이급SDS-PAGE검측NTR재Hela세포중적표체,MTT법검측NTR/CB1954대Hela세포활력적영향,류식세포술검측아이배체세포솔개변,PI/Hoechest33258쌍염형광현미경하관찰Hela세포조망솔.결과: 성공구건료진핵표체재체pcDNA3-nfsB,획득은정표체NTR적Hela세포주,재mRNA수평이급단백수평검측도NTR재Hela세포중적표체,NTR/CB1954자살기인계통명현영향Hela세포적활력,증가료Hela세포적조망솔.결론: NTR/CB1954자살기인계통대Hela세포재체외통과조망산생명현적살상효응.
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.