目的 探讨六味地黄丸主要入血成分对自杀基因系统(HSV-tk/GCV)杀伤黑色素瘤B16细胞的增效作用及作用机制。方法 采用MTT法检测六味地黄丸主要入血成分丹皮酚、马钱素、莫诺苷、5-羟甲基-2-糠酸和獐牙菜苷5种化合物及其混合组分对HSV-tk/GCV杀伤B16细胞的影响；入血成分混合组分处理脾淋巴细胞后培养上清对HSV-tk/GCV杀伤B16细胞的影响；荧光显微镜观察及流式细胞术结合荧光示踪法分析含药淋巴细胞上清对细胞间缝隙连接通讯(GJIC)功能的影响及Western blot分析缝隙连接蛋白(Cx43)表达；MTT法检测含药淋巴细胞上清对HSV-tk/GCV杀伤B16细胞的增敏作用。结果 HSV-tk/GCV联合丹皮酚、马钱素、莫诺苷、5-羟甲基-2-糠酸和獐牙菜苷5种化合物及5种化合物的混合组分，均未显示其对自杀基因的杀伤有直接增效作用；而混合组分处理脾淋巴细胞后培养上清对HSV-tk/GCV杀伤B16细胞具有协同增效作用，并呈一定的量效关系。含药淋巴上清可明显促进Cx43表达，改善B16细胞间缝隙通讯功能。含药淋巴上清对HSV-tk/GCV的B16细胞杀伤敏感性则无明显影响。结论 六味地黄丸主要入血成分可能刺激脾淋巴细胞产生某些活性物质，并对HSV-tk/GCV杀伤B16细胞起增效作用，这些淋巴细胞产生的免疫活性物质可能才是六味地黄丸对肿瘤自杀基因疗法增效作用的药效物质基础；其增效机制可能与改善B16细胞间GJIC相关。

Objective To explore the synergistic effect and mechanism of plasma constituents after oral administration of Liuwei Dihuang Pills(LDP) in suicide gene killing of B16 cells. Methods MTT assay was adopted to observe the effect of plasma constituents after oral administration of LDP，paeonol，loganin，morroniside，5-hydroxymethyl-2- furoic acid，and sweroside，and their mixture composition on HSV-tk/GCV suicide gene system in killing B16 cells. We also observed the effects of culture supernatant of lymphocytes treated by the mixture of five plasma components on HSV-tk/GCV suicide gene system in killing B16 cells. The gap junction function was analyzed by Fluorescent tracer with flow cytometry and fluorescence microscopy，and the expression of connexin was detected by Western blotting. The sensitization effects of drug-treated lymphocytes supernatant on tk/GCV system of B16-tk+ cells were investigated by MTT assay. Results Not only the 5 components but also their mixture compounds did not show the bystander effect when they were used together with tk/GCV system. However，the drug-treated lymphocytes supernatant showed the synergism in dose-effect relationship. Drug-treated lymphocytes supernatant significantly enhanced Cx43 expression and improved the GJ function of B16 cells，but had no significant effect on the sensitization of B16-tk cells to tk/GCV system. Conclusion Plasma LDP constituents can stimulate the lymphocytes to produce certain active substances which enhance the TSV-tk/GCV bystander effect on B16 cells. The immune active substances may be the material basis of the synergism of LDP in promoting suicide gene therapy for tumor，and the mechanism is probably associated with the improvement of B16 cells GJ function.

国家自然科学基金资助项目(81072906,30973811)。