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目的观察超声微泡造影剂携单纯疱疹病毒Ⅰ型胸昔激酶(HSV1-TK)基因体外转染及联合前药更昔洛韦(GCV)对前列腺癌细胞株PC-3的抑制效应。方法超声辐照PC-3细胞,MTT筛选出最适辐照时间;将含有HSV1-TK基因的质粒通过静电吸附在脂质微泡表面,采用最适超声辐照转染,并用荧光显微镜及Western blot观察TK基因的转入及表达;MTT法观察使用不同浓度的前药更昔洛韦(GCV)后细胞增殖抑制率;流式细胞仪检测最适浓度的前药GCV对转染后的前列腺癌细胞PC-3的周期的影响;MTT法观察HSV1-TK/GCV自杀基因系统对前列腺癌PC-3细胞杀伤效应。结果 MTT检测显示超声强度为1.0 W/cm2,频率为1 MHz,辐照时间30 s时超声辐照对细胞无明显的抑制作用;通过超声辐照后HSV1-TK基因可以顺利转入PC-3细胞中并有稳定的表达。与对照组相比,当前药GCV浓度在100μg/ml时,超声+微泡+TK组细胞杀伤率明显高于其他各组,细胞存活率约32%,低于其他各组(P<0.05);流式细胞术检测显示大多数细胞被阻断在S期,细胞生长受到明显抑制。结论以超声辐照微泡造影剂为载体转染自杀基因联合前药GCV,对人前列腺癌细胞有明显杀伤作用。
Objective To observe the inhibitory effect of ultrasound microbubble contrast agent (HSV1-TK) transfected HSV1-TK gene in vitro and its effect on prostate cancer cell line PC-3 by ganciclovir (GCV). METHODS: PC-3 cells were irradiated with ultrasound and the optimal irradiation time was selected by MTT. Plasmids containing HSV1-TK gene were electrostatically adsorbed on the surface of lipid microbubbles, and were transfected by optimal ultrasound irradiation. Fluorescence microscopy and Western blotting was used to observe the transfection and expression of TK gene. MTT assay was used to observe the inhibitory rate of cell proliferation with different concentration of ganciclovir (GCV); GCV was used to detect the optimal concentration of prodrug The effect of HSV1-TK / GCV suicide gene system on the PC-3 cell cycle was investigated by MTT assay. Results MTT assay showed that ultrasound irradiation at 1.0 W / cm2, frequency 1 MHz and irradiation time 30 s had no obvious inhibitory effect on the cells. HSV1-TK gene could be transferred into PC-3 Cells and stable expression. Compared with the control group, the cell killing rate of ultrasound + microbubble + TK group was significantly higher than that of other groups when the concentration of GCV was 100μg / ml, the cell survival rate was about 32%, which was lower than other groups (P <0.05) Flow cytometry showed that most cells were blocked in S phase and cell growth was significantly inhibited. Conclusion Ultrasound irradiated microbubble contrast medium transfected with suicide gene combined with prodrug GCV has a significant killing effect on human prostate cancer cells.