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24 h,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,Western blot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、1000.0 mg/L共8个浓度梯度的前药更昔洛韦(GCV),72 h后MTr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tk mRNA相对表达量为1.13,是其余各组的7～9倍,Western blot 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50 mg/L以上培养72 h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.

abstractsObjective 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-EGFP-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 fluorescence 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 36.00％ respectively.TK protein in group E was higher than other groups ( P ＜ 0.05 ).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-targeted 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.