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目的:探讨六味地黄丸对大鼠肝癌CBRH7919细胞Cx26表达的影响,初步阐明其对自杀基因治疗的增效作用是否与缝隙连接机制相关。方法:应用血清药理学研究方法制备六味地黄丸含药血清。采用MTT法检测六味地黄丸含药血清联合单纯疱疹病毒-胸苷激酶/丙氧鸟苷(HSV-tk/GCV)自杀基因系统治疗的杀伤效应:应用蛋白印迹(Western blot)技术、间接免疫荧光法及荧光定量逆转录-聚合酶链反应(RT-PCR)技术检测其对CBRH7919细胞株Cx26蛋白及mRNA表达的影响。结果:①六味地黄丸含药血清联合自杀基因治疗组肝癌细胞存活率明显低于单纯自杀基因组(10%tk+/GCV加对照血清)(P<0.01),且联合作用均具有协同性(Q>1.15);②Western blot检测显示六味地黄丸含药血清能提高Cx26蛋白的表达,并有明显的量效关系;③间接免疫荧光法(FITC)法共聚焦显微镜观测显示近似的结果:蛋白定位于细胞膜和胞浆,尤其在细胞膜的表达明显增多,含药血清组的Cx26蛋白荧光强度比对照组明显增加(P<0.05)。结论:六味地黄丸增强自杀基因旁杀伤效应的机制与其促进肝癌细胞Cx26 mRNA及蛋白表达,增加膜上定位表达进而改善细胞间连接通讯(GJIC)功能有关。
Objective: To investigate the effect of Liuweidihuangwan on the expression of Cx26 in rat hepatoma CBRH7919 cells and to clarify whether its synergistic effect on suicide gene therapy is related to the gap junction mechanism. Methods: Serum pharmacology study method was used to prepare Liu Wei Di Huang Wan containing serum. MTT assay was used to determine the killing effect of Liuweidihuangwan combined with HSV-tk / GCV suicide gene system. Western blotting, indirect immunofluorescence Method and fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) technology to detect the expression of Cx26 protein and mRNA in CBRH7919 cell line. Results: ① The survival rate of hepatoma cells in Liuweidihuangwan combined with suicide gene therapy group was significantly lower than that in suicide gene only group (10% tk + / GCV plus control serum) (P <0.01), and the combined effects were both synergistic (Q> 1.15) .②Western blot showed that the drug-containing serum of Liuweidihuangwan could improve the expression of Cx26 protein and had a significant dose-response relationship. (3) Confocal microscopy with indirect immunofluorescence (FITC) method showed similar results. The protein localized in the cell membrane And cytoplasm, especially in the cell membrane. The fluorescence intensity of Cx26 protein in the drug-containing serum group was significantly higher than that in the control group (P <0.05). Conclusion: The mechanism of Liuweidihuang Pills in enhancing the para-lethal effect of suicide gene is related to promoting the expression of Cx26 mRNA and protein in hepatocellular carcinoma cells, increasing the localization of membrane and improving the function of GJIC.