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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 investigate the synergic effect and mechanism of the main components of Liuweidihuang Pill on suicide gene system (HSV-tk / GCV) killing melanoma B16 cells. Methods MTT assay was used to detect the inhibitory effects of five compounds including paeonol, loganin, morroniside, 5-hydroxymethyl-2-furoic acid and swertia glycosides of Liuweidihuangwan on HSV- tk / GCV on B16 cells. The effects of culture supernatants of HSV-tk / GCV-killed B16 cells after treatment of splenic lymphocytes with mixed components of infiltrating components were observed by fluorescence microscopy and flow cytometry combined with fluorescent tracer assay The effects of drug-loaded lymphocyte supernatant on the function of GJIC and the expression of connexin (Cx43) by Western blot were detected by MTT assay. The sensitivities of drug-containing lymphocyte supernatant to HSV-tk / GCV-killed B16 cells were determined by MTT assay effect. Results The five components of HSV-tk / GCV combined with paeonol, loganin, morroniside, 5-hydroxymethyl-2-furoic acid and swertia glycosides and the mixed components of 5 compounds did not show that The killing effect of suicide gene had a direct synergistic effect. The mixed culture supernatant of spleen lymphocytes had a synergistic effect on HSV-tk / GCV killing B16 cells, and showed a dose-effect relationship. Drug-containing lymphatic supernatant can significantly promote the expression of Cx43 and improve the B16 cell gap communication. Drug-containing lymph supernatant of HSV-tk / GCV B16 cell killing sensitivity had no significant effect. Conclusion The main components of Liuweidihuang Pills may stimulate the spleen lymphocytes to produce some active substances and enhance the cytotoxicity of HSV-tk / GCV against B16 cells. The immunocompetent substances produced by these lymphocytes may be the effect of Liuweidihuangwan on tumor Suicide gene therapy synergistic effect of the material basis; its synergistic mechanism may be related to the improvement of B16 intercellular GJIC.