六味地黄丸对自杀基因系统治疗黑色素瘤的增效作用

Synergistic Action of Liuwei Dihuang Bolus in the Treatment of Murine Transplanted Melanoma with HSV-tk/GCV System

【目的】研究六味地黄丸对HSV1-tk/GCV自杀基因系统治疗黑色素瘤的增效作用,以期探讨建立中西医结合肿瘤自杀基因联合治疗方案的可行性。【方法】培养病毒包装细胞PT67/tk,病毒上清感染黑色素瘤细胞株B16,G418筛选2周,获得抗性细胞克隆,命名为B16/tk并进行体外GCV杀伤试验;证明具有杀伤活性后,将B16/tk与野生型B16按1∶4的比例混合后接种于C57BL/6J小鼠右腋下皮下组织内,以复制小鼠移植性黑色素瘤模型。将复制模型的小鼠分为模型对照组、六味地黄丸组、自杀基因tk/GCV治疗组和联合治疗组(N=11),六味地黄丸从接种第2天起治疗25 d,tk/GCV从接种第7天起治疗20 d,另设正常对照组观察疗效。【结果】体外杀伤实验显示,GCV对B16/tk肿瘤细胞具有明显的杀伤效应,而对野生型B16肿瘤细胞无明显的杀伤作用,表明体外病毒感染黑色素瘤细胞成功,病毒携带的外源性自杀基因已表达且具有生物学活性。联合治疗(自杀基因联合六味地黄丸)对小鼠移植性黑色素瘤生长具有明显抑制作用,联合治疗组肿瘤质量与模型对照组比较差异具有统计学意义(P<0.05),其抑瘤率达到87.1%;而单纯自杀基因组和六味地黄丸组肿瘤质量与模型对照组比较差异无统计学意义(P>0.05),抑瘤率分别为62.8%和54.4%。病理检查结果显示,肉眼见治疗组肿瘤体积减小,以联合治疗组更明显。光镜下见治疗组肿瘤细胞密度相对较低,肿瘤周围有较多纤维结缔组织增生及炎症细胞浸润,其中联合治疗组更明显。结果表明:自杀基因联合六味地黄丸治疗对小鼠移植性黑色素瘤生长具有抑制作用,其疗效优于单纯自杀基因治疗或单纯六味地黄丸治疗。【结论】六味地黄丸可能通过自杀基因疗法旁杀伤效应的免疫机制或其他机制对肿瘤自杀基因疗法起到增效作用。该研究工作初步证明了建立自杀基因联合中医药疗法的中西医结合肿瘤基因治疗方案具有可行性。

[ Objective] To study the synergistic action of Liuwei Dihuang Bolus （LDB） on murine transplanted melanoma with Herpes simplex virus thymidine kinase/ganciclovir-mediated （HSV-tk/GCV） system and to investigate the feasibility of establishing TCM-WM suicide gene therapy for the treatment of tumor. [ Methods] Melanoma cell strain BI6 was transfected with the recombinant retrovirus in the supematant of virus-producing packaging cell line PT67/tk culture. After transfection, melanoma cell strain B16 was cultured with G418 for 2 weeks to obtain the clone of antiG418 tk cell, named as B16/tk. The killing activity of B16/tk was tested by in-vitro GCV test. After then, the mixture of B16/tk with killing activity and B16 wild type at the proportion of 1：4 were subcutaneously implanted into the right axillae of C57BL/6J mice to establish models of murine transplanted melanoma. The mice models were divided into 4 groups： model group, LDB group, suicide gene tk/GCV group and combined therapy group, 11 mice in each group. Ten mice were in the normal group serving as the control. LDB treatment began in the 2^nd day of implantation and lasted 25 days, and suicide gene tk/GCV treatment began in the 7^nd day of implantation and lasted 20 days. After treatment, the therapeutic effect was evaluated in the different groups. [Results] The results of in-vitro GCV test showed that GCV had an obvious killing effect on Bl6/tk but had no killing effect on B16 wild type, indicating that melanoma cell strain BI6 was infected with virus in vitro successfully and the exogenous suicide gene carried by the virus had an bioactivity. The combined therapy of LDB and suicide gene tk/GCV had an obviously inhibitory effect on the growth of murine transplanted melanoma, and the difference of tumor mass weight was significant between the model group and the combined therapy group （P 〈 0.05）. The tumor-inhibitory rate was 87.1% in the combined therapy group, which did not differ from 62.8% in suicide gene tk/GCV group or from 54.4% in LDB group （P 〉 0.05）. The results of macroscopic examination showed that the tumor mass became smaller, in particular in the combined therapy. The results under light microscope showed that the density of tumor cells was lowered, and the proliferation of fibro-connective tissues around the tumor cells, as well as the infiltration of inflammatory cells was obvious, particularly in the combined therapy group. [Conclusion] The combined therapy of LDB and suicide gene tk/GCV has an better inhibitory effect on the growth of murine transplanted melanoma than LDB or suicide gene tk/GCV only. The synergistic mechanism of LDB may be related to the immuno-regulation of bystander effect of suicide gene therapy. The results of this study preliminarily prove the feasibility of establishing TCM-WM suicide gene therapy for the treatment of tumor.