腺病毒介导HSV-TK/GCV自杀基因系统靶向性治疗前列腺癌实验研究  

The selective killing effect of adenoviral mediated HSV-TK/GCV suicide gene system on prostate cancer cells

作　　者：白志杰[1] 黎玮[1] 王亚轩[1] 张勇[1] 马洪顺[1] 

机构地区：[1]天津市第一中心医院泌尿外科,天津300192

出　　处：《现代泌尿外科杂志》2009年第6期447-450,共4页Journal of Modern Urology

摘　　要：目的观察腺病毒介导单纯疱疹病毒胸苷激酶基因/丙氧鸟苷(HSV-TK/GCV)自杀基因系统在人端粒酶逆转录酶(hTERT)启动子调控下对人前列腺癌细胞的靶向性体外杀伤效应。方法利用不同感染复数(MOI)的重组腺病毒携带增强型绿色荧光蛋白(EGFP)基因感染前列腺癌细胞LNCaP和人成纤维细胞MRC-5,荧光显微镜下观察其感染效率;利用携带不同启动子的重组腺病毒Ad-hTERT-HSV/TK以及Ad-CMV-HSV/TK感染LNCaP和MRC-5细胞,加入不同浓度GCV,MTT法观察受转染细胞的存活率。结果重组腺病毒Ad-hTERT-EGFP能特异地转染LNCaP,其转染效率随重组病毒的MOI增加而升高(P<0.01),MOI为1时转染率为8.3%,MOI为1 000时转染率达100%;应用GCV处理后,Ad-CMV-HSV/TK对LNCaP和MRC-5细胞均有杀伤作用,而Ad-hTERTp-HSV/TK只杀伤LNCaP(P<0.001),随着MOI和GCV浓度的增加,LNCaP细胞存活率明显降低(P<0.01),MOI为1和GCV浓度为1μmol/L时存活率为95.4%,MOI为100和GCV浓度为1 000μmol/L时存活率仅为6.1%,并有旁观者效应。结论重组腺病毒携带EGFP报告基因可准确、简便地确定转染效率;hTERT启动子调控的重组腺病毒介导的HSV-TK/GCV自杀基因系统对人前列腺癌细胞有靶向杀伤作用。Objective To observe the selective killing effect of adenoviral mediated herpes simplex virus-thymidine kinase/ganciclovir （HSV-TK/GCV） suicide gene system controlled by human telomerase reverse transcriptase （hTERT） promoter on prostate cancer cells in vitro. Methods Prostate cancer cell line LNCaP and human fibroblast cell line MRC-5 were transfected by the recombinant adenovirus of different multiplicities of infection （MOI）, and the infection rate was measured by observing the expression of enhanced green fluorescent protein （EGFP） under the fluorescent microscopy. Ad-hTERT-HSV/TK and Ad-CMV-HSV/TK were transfected into LNCaP and MRC-5 followed by GCV treatment. The relative survival rate of cells in presence of predrug GCV was measured with MTT method. Results Recombinant adenovirus Ad- hTERT-EGFP could selectively infect LNCaP cells, with the infection rate associated with the MOI of recombinant adenovirus （P〈0.01）. When MOI was 1, the infection rate was 8.3%. When MOI was 100, the infection rate was 100%. GCV inhibited significantly the growth of both cell lines infected with Ad-CMV-HSV/TK and the growth of LNCaP infected with Ad-hTERT- HSV/TK （P〈0.001）. The relative survival rate of LNCaP was correlated to both the concentration of the predrugs and the MOI of recombinant adenovirus （P〈〈0.01）. When MOI was 1, 1μmol/L GCV killed 95.4% of LNCaP. When MOI was 100, 1000μmol/L GCV killed 6.1% of LNCaP. Conclusion Recombinant adenovirus vector containing EGFP can report the infection rate correctly. Adenoviral vector containing hTERT promoter for the HSV/TK plus GCV treatment appears to be highly selective in killing prostate cancer cell line in vitro.

关 键 词：前列腺癌 重组腺病毒 自杀基因 人端粒酶逆转录酶启动子 

分 类 号：R697[医药卫生—泌尿科学]