超声微泡介导HSV1-TK／GCV自杀基因系统对人肝癌细胞株HepG2的杀伤作用  

Ultrasound contrast agent-mediated HSV1-TK/GCV suicide gene system for hepatocellular carcinoma in vitro

作　　者：唐勇[1] 何建峰[1] 周世骥[2] 刘长安[2] 龚建平[2] 刘作金[2] 

机构地区：[1]重庆黔江中心医院肝胆外科,409000 [2]重庆医科大学附属第二医院肝胆外科

出　　处：《中华实验外科杂志》2011年第10期1653-1655,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872977）

摘　　要：目的观察超声微泡（UM）介导HSV1-TK／GCV自杀基因系统对人肝癌细胞株HepG2的杀伤效率。方法将HepG2接种在培养板中，随机分为以下5组：（A）空白对照组；（B）单纯质粒组；（c）超声微泡＋质粒组；（D）超声＋质粒组；（E）超声＋超声微泡＋质粒组。转染后24h，荧光显微镜观察各组细胞绿色荧光的表达，流式细胞仪检测各组细胞的转染效率，噻唑蓝（MTT）比色法检测转染方法对HepG2活性的影响，Westernblot法检测各组细胞的TK蛋白表达；转染后24h，各组细胞加入0．0．1、1．0、10．0、50．0、100．0、500．0、1000．0mg／L共8个浓度梯度的前药更昔洛韦（GCV），72h后MTY法检测各组细胞的存活率。结果荧光显微镜下E组的绿色荧光强度明显高于其他各组；流式细胞仪检测基因转染率分别为0％、0．39％、0．61％、1．10％、36．00％，荧光定量聚合酶链反应（PCR）检测提示E组tkmRNA相对表达量为1．13，是其余各组的7～9倍，Westernblot条带灰度分析显示E组TK蛋白的明显高于其他各组（P〈0．05）；转染方法对HepG2细胞活性无明显影响（P〉0．05）；GCV浓度在50mg／L以上培养72h后，低频超声＋超声微泡＋质粒组细胞几乎全部被杀灭，存活率为0，杀伤效应最强（P〈0．05），其余各组几乎无杀伤作用。结论超声破坏微泡造影剂可提高HSV1-TK基因在HepG2中的转染和表达，增强HSV1-TK／GCV自杀基因系统对肝癌细胞的杀伤效应，而超声辐照和微泡造影剂在转染过程中对细胞的活性无影响。Objective To explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble （UM） destruction mediated by HSV1-TK/GCV suicide gene system, and indentify the effects of UM destruction treatment on the cell viability. Methods HepG2 cells were seeded in the 24-well plates, and randomly divided into 5 groups ： （A） blank control group ; （B） HSV1-TK： pIRES2-EGFP-TK only; （C） HSV1-TK ＋ UM： gene and ultrasound contrast agent; （D） HSV1-TK ＋ US ： gene and UM ＋ pulsed-ultrasound; （E） HSV1-TK ＋ UM ＋ US ： gene ＋ US ＋ pIRES2-EG- FP-TK ＋ pulsed-ultrasound. After 24 h, the expression of EGFP was observed under the fluorescent microscopy and quantified by flow cytometry. Western Blotting was used to detect the expression of TK protein. Twenty-four h after transfection, different concentrations of GCV （0, 0. 1, 1.0, 10. 0, 50. 0, 100. 0, 500. 0, 1000. 0 mg/L） were added into the culture medium, then cells were cultured for another 72 h. The viability of HepG2 cells was measured by methyl thiazol tetrazolium （MTT） assay. Results Gene fluores- cence intensity and tansfection efficiency in group E were higher than other groups. The gene transfection efficiency in groups A, B, C, D and E was 0%, 0. 39%, 0. 61%, 1.10% and 56. 00% respectively. TK protein in group E was higher than other groups （P 〈 O. 05）. The cell viability had no significant difference among group before addition of GCV. Almost all HepG2 cells in group E were killed when the concentration of GCV was above 50 mg/L, but almost no killing effect was observed in other groups after addition of different concentrations of GCV. Conclusion Ultrasound-tazgeted microbubble destruction can enhance the efficiency of gene transfection without obvious damage to cell viability in HepG2 cells. The method can also enhance the killing effect of HSV1-TK/GCV suicide gene system in vitro.

关 键 词：癌 肝细胞 超声 造影剂 HSV1-TK/GCV 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]