人甲胎蛋白增强子驱动自杀基因靶向杀伤肝癌细胞的实验研究  

Suicide gene driven by human alpha fetoprotein enhancer kills hepatocellular carcinoma cells

作　　者：朱宝和[1] 王成友[1] 倪勇[1] 张敏杰[1] 廖允军[1] 詹勇强[1] 韩庆[1] 

机构地区：[1]深圳市第二人民医院普通外科,518030

出　　处：《中华消化外科杂志》2011年第4期274-277,共4页Chinese Journal of Digestive Surgery

基　　金：国家高技术研究发展计划（863计划）项目（2007AA021809）;深圳市科技计划项目（200801005）

摘　　要：目的探讨人AFP增强子驱动的单纯疱疹病毒胸苷激酶（HSV-TK）／丙氧鸟苷自杀基因系统体内外靶向杀伤肝癌细胞的效应。方法构建人AFP增强子驱动的pAFP-cDNA3．1-TK自杀基因真核表达质粒；利用脂质体将质粒转染入AFP阳性肝癌细胞株HepG2和AFP阴性肝癌细胞株SMMC7721；采用RT-PCR和Westernblot检测TKmRNA和蛋白表达；MTr检测细胞的存活率；观察丙氧鸟苷对肝癌细胞体外增殖、生长曲线和细胞凋亡的作用，以及丙氧鸟苷体内抑制肿瘤生长的效应。采用t检验分析相关数据。结果成功构建pAFP-cDNA3．1-TK自杀基因真核表达质粒并转染入肝癌细胞；AFP阳性肝癌细胞株HepG2中能检测到TKmRNA和蛋白表达；丙氧鸟苷呈剂量和时间依赖性抑制转染后肝癌细胞株HepG2的生长并诱导其凋亡；AFP阴性肝癌细胞株SMMC7721中没有TKmRNA和蛋白表达，细胞增殖、生长曲线和凋亡均不受影响，两者比较，差异有统计学意义（t：2．58，2．73，3．12，P〈0．05）。丙氧鸟苷可以特异性地抑制转染后肝癌细胞株HepG2治疗组中肿瘤的生长，肿瘤抑制率为46％，而在转染后肝癌细胞株SMMC7721治疗组中，对肿瘤生长无明显抑制作用，两者比较，差异有统计学意义（t=3．36，P〈0．05）。结论人AFP增强子驱动的HSV-TK／丙氧鸟苷自杀基因系统可以靶向杀伤AFP阳性肝癌细胞，抑制肿瘤生长。Objective To investigate the effect of herpes simplex virus thymidine kinase/ganciclovir （HSV-TK/GGV） system driven by human alpha fetoprotein （AFP） enhancer on hepatocellular carcinoma （HCC） cells in vitro and in vivo. Methods HCC-specific eukarotypic expression vector carrying suicide gene driven by AFP enhancer （pAFP-cDNA3.1-TK） was constructed. The plasmid was trasfected to AFP-positive HepG2 cells and AFP-negative SMMC7721 cells by liposomes. Protein and mRNA expressions of TK were detected by RT-PCR or Western blot. The survival rates of HCC ceils were detected by methyl thiazolyl tetrazolium assay. The effects of GGV on the in vitro proliferation, survival and apoptosis of HCC cells were observed, and the inhibitive effect of GGV on the survival of HCC cells in vivo was also detected. All data were analyzed by using the t test. Results The pAFP-cDNA3.1-TK was successfully constructed and transfected to the HCC cells. The protein and mRNA expressions of TK were detected in AFP-positive HepG2 cells. GGV dose- and time-dependently inhibited the growth and induced the apoptosis of HepG2 ceils in vitro, but it had no effect on SMMC7721 ceils. No protein or mRNA expression of TK was detected in the SMMC7721 cells. There was a significant difference on the inhibitory effects of GGV on HepG2 cells and SMMC7721 ceils （t = 2. 58, 2. 73, 3. 12, P 〈 0.05）. GGV specifically inhibi-ted the growth of AFP-positive HepG2 cells, and the inhibition rate was 46%; the growth of AFP-negative SMMC7721 cells was not influenced by GGV. There was a significant difference in the inhibitive effect of GGV on the growth of HepG2 ceils and SMMC7721 cells （ t= 3.36, P 〈 0.05）. Conclusion HSV-TK/GGV system driven by human APF enhancer kills APF-positive HCC ceils and inhibits the growth of I-ICC ceils.

关 键 词：肝肿瘤 基因疗法 自杀基因 

分 类 号：R735.7[医药卫生—肿瘤]