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【目的】探讨六味地黄丸含药血清对单纯疱疹病毒核苷激酶/丙氧鸟苷(HSV-tk/GCV)系统杀伤小鼠黑色素细胞瘤B16细胞株的增效作用,为提高临床基因治疗肿瘤疗效提供新思路。【方法】构建并鉴定恶性黑色素瘤B16细胞HSV-tk/GCV治疗系统,制备六味地黄丸含药血清及对照血清,将B16、B16/tk细胞按20%(1:4)的比例混合制成细胞悬浮液接种于96孔培养板上,分为对照血清组、含药血清组、对照血清联合GCV组、含药血清联合GCV组,其中含药血清组及含药血清联合GCV组按血清体积分数计又分高(10%)、中(5%)、低(2.5%)3组,GCV的终浓度为25μmol/L,按分组添加相应的含药血清,每孔培养液总体积200μL,37℃、体积分数5%的CO2培养箱培养。48h后采用四甲基偶氮唑盐(MTT)法检测杀伤效应。采用金正钧Q值法分析六味地黄丸含药血清与自杀基因治疗系统联合作用对旁观者效应是否具有协同性。【结果】MTT实验表明联合用药组对B16细胞的抑制率明显高于其他组,六味地黄丸含药血清对B16细胞株HSV-tk/GCV系统的增效作用存在量效关系:2.5%、5%、10%含药血清联合20%tk/GCV组的实际抑制率(48.75%、59.39%、69.28%)显著高于理论抑制率(38.13%、38.67%、39.92%),金正钧Q值分别为1.28、1.54、1.74,均大于1.15,具有协同增效作用。【结论】六味地黄丸含药血清对B16细胞株HSV-tk/GCV系统旁观者效应有明显的增效作用,且具有一定的量效关系。
【Objective】 To investigate the synergistic effect of Liuwei Dihuang Pill containing serum on HSV-tk / GCV system killing mouse melanocytoma B16 cell line, in order to improve clinical gene therapy of tumor Efficacy provides new ideas. 【Method】 HSV-tk / GCV treatment system was constructed and identified in malignant melanoma B16 cells. The serum and control serum of Liuweidihuangwan was prepared. The B16 and B16 / tk cells were mixed in the ratio of 20% (1: 4) The cell suspension was inoculated into 96-well culture plates and divided into control serum group, drug-containing serum group, control serum group and GCV group, drug-containing serum and GCV group. The drug-containing serum group and drug- The scores were divided into high (10%), medium (5%) and low (2.5%) groups. The final GCV concentration was 25μmol / L. The corresponding drug- 37 ℃, volume fraction of 5% CO2 incubator culture. Forty-eight hours later, the killing effect was detected by MTT assay. Jin Zhengjun Q value analysis of Liuwei Dihuang pill containing serum and suicide gene therapy system on the bystander effect whether there is synergy. 【Results】 The results of MTT assay showed that the inhibition rate of B16 cells in combination group was significantly higher than that in other groups. The synergistic effect of Liuwei Dihuang Pill containing serum on HSV-tk / GCV system was 2.5%, 5 (48.75%, 59.39%, 69.28%) in the 10% drug-serum combined with 20% tk / GCV group were significantly higher than the theoretical inhibition rates (38.13%, 38.67%, 39.92% 1.28,1.54,1.74, both greater than 1.15, with synergy. 【Conclusion】 Liuwei Dihuang Pill containing serum has a synergistic effect on bystander effect of HSV-tk / GCV system on B16 cells and has a certain dose-effect relationship.