六味地黄丸增强黑色素瘤自杀基因系统旁观者效应的体外研究  

Enhancement of Liuwei Dihuang Bolus on Bystander Effect of HSV-tk/GCV Suicide Gene Therapy for Mice Melanoma Cells

作　　者：易华[1] 郭玉荣[2] 杜标炎[1] 谭宇蕙[1] 吴映雅[1] 

机构地区：[1]广州中医药大学基础医学院,广东广州510405 [2]广西柳州市中医院,广西柳州545001

出　　处：《广州中医药大学学报》2010年第5期498-502,556,557,共7页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(编号:30572433)

摘　　要：【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 bystander effect of medicated serum of Liuwei Dihuang Bolus （LDB） on mice melanoma B16 cells treated by HSV-tk/GCV treatment system. Methods We constructed and identified B16 cells HSV-tk/GCV treatment system. Medicated serum was obtained from SD rats which were treated with LDB, and non-medicated serum was obtained from rats receiving normal saline. The cell suspension mixed with B16 and B16/tk at the ratio of 1：4 were implanted in 96-well culture plate, and were divided into control serum group, LDB serum group , GCV ＋ control serum group, LDB serum ＋ GCV group, and combination serum group according to the culture serum added, and the total volume of the culture solution in each well was 200μL. The combination serum group was divided into three subgroups （ volume fraction being 10. 0 % , 5.0% and 2. 5% respectively）. The final concentration of GCV was 25μmol/L. The cells were cultured in 5% CO2 incubator at 37℃. After culturing for 48 hours, methyl thiazolyl tetrazolium （MTT） assay was used to detect the killing effect. The synergistic action of LDB serum and HSV-tk/GCV treatment system on bystander effect was evaluated by Jinzheng]un Q value. Results The results of MTT test showed that the combination serum group had a higher inhibitory rate for B16 cells than other groups. The enhancement of LDB on the bystander effect of HSV-t/GCV was in a dose-effect manner. LDB serum 2.5%, 5.0% and 10. 0% combined with 20% tk/GCV had the actual inhibitive rate of 48.75%, 59.39% and 69.28% , respectively, significantly higher than the theoretical inhibitive rate （38.13%, 38.67% and 39.92% respectively）. Jinzhengjun Q value was 1.28, 1.54, 1.74 （all exceeding 1.15）, respectively, indicating the good synergistic action of LDB serum. Conclusion Medicated serum of LDB has obvious synergistic bystander effect on HSV-tk/GCV treatment system for mice melanoma B16 cells, in a doseeffect manner.

关 键 词：六味地黄丸/药理学 自杀基因疗法 黑色素细胞/病理学 细胞培养 

分 类 号：R285.5[医药卫生—中药学]