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目的:探讨六味地黄丸主要入血成分对单纯疱疹病毒胸苷激酶/丙氧鸟苷(HSV-tk/GCV)自杀基因系统杀伤大鼠肝癌细胞(CBRH7919)的增效作用。方法:按CBRH7919/tk占总细胞数10%的比例混合CBRH7919与CBRH7919/tk细胞,分别接种96孔板,每孔加细胞悬液100μL(2×103个/孔),设置对照组、低、中、高3种浓度的六味地黄丸入血成分组、GCV组、各浓度六味地黄丸入血成分联合GCV组;培养24 h后分别加入入血成分马钱素和丹皮酚(质量浓度均为25,50,100μmol.L-1),莫诺苷、5-羟甲基-2-糠酸、獐牙菜苷(浓度均为37.5,75,150μmol.L-1)5种化合物和5种化合物混合组分(质量浓度为0.006 7,0.013 5,0.027 g.L-1);及其混合组分(质量浓度为0.034 g.L-1)作用后淋巴细胞培养上清(占培养液体积分数7.5%,15%,30%),设6个复孔,37℃,5%CO2培养箱培养72 h后MTT法测定细胞存活率,检测六味地黄丸主要入血成分及淋巴细胞培养上清对自杀基因系统杀伤肝癌细胞的影响。结果:自杀基因系统联合马钱素、莫诺苷、5-羟甲基-2-糠酸、獐牙菜苷和丹皮酚5种化合物及5种化合物的混合组分,均未显示其对肿瘤自杀基因疗法的直接增效作用;但含药上清联合GCV组对肝癌细胞生长抑制率分别为(22.72±3.12)%,(26.68±1.92)%,(30.50±3.53)%;与单独自杀基因(10%tk/GCV)组抑制率(15.37±3.96)%比较,差异均有统计学显著性意义;q值分别为1.50,1.75,2.24,均大于1.15,具有协同增效作用,并呈一定的量效关系。结论:六味地黄丸主要入血成分能刺激淋巴细胞产生某些活性物质,并发挥了对HSV-tk/GCV自杀基因治疗系统对肝癌CBRH7919细胞杀伤的增效作用,这些淋巴细胞产生的内源性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。
Objective: To investigate the synergistic effects of Liuweidihuang Pill main blood components on HSV-tk / GCV suicide gene system-killing rat hepatoma cells (CBRH7919). METHODS: CBRH7919 and CBRH7919 / tk cells were mixed with CBRH7919 / tk at a ratio of 10% of the total cells. The cells were inoculated into 96-well plates, and 100 μL (2 × 103 cells / well) of cell suspension was added into each well to set the control group, Medium and high concentrations of Liuweidihuangwan into the blood component group, GCV group, the concentration of Liuweidihuangwan into the blood component GCV group; cultured for 24 h were added into the blood components of loganin and paeonol (mass concentration (25, 50, 100 μmol·L-1), 5 compounds of morroniside, 5-hydroxymethyl-2-furoic acid and swertia glycosides (37.5,75 and 150μmol.L-1 in concentration) (Concentration of 0.006 7, 0.013 5, 0.027 gL-1) and the mixed lymphocyte culture supernatant (7.5%, 15 %, 30%). Six replicate wells were cultured at 37 ℃ in 5% CO2 incubator for 72 h. MTT assay was used to determine the cell viability. The main components of the Liuweidihuangwan injection and the supernatant of the lymphocyte culture were selected to kill the suicide gene system Effect of liver cancer cells. Results: The combination of sumatran system, loganin, morroniside, 5-hydroxymethyl-2-furoic acid, swertin and paeonol, and the mixed components of 5 compounds did not show any significant difference (22.72 ± 3.12)%, (26.68 ± 1.92)%, (30.50 ± 3.53)%, respectively, in GCV group and drug-induced supernatant combined with GCV. Compared with suicide alone (10% tk / GCV) group (15.37 ± 3.96)%, the differences were statistically significant; q values were 1.50,1.75,2.24, were greater than 1.15, with synergistic effect, and was A certain amount of effective relationship. Conclusion: The main components of Liuweidihuang Pills can stimulate certain active substances in lymphocytes and play a synergistic effect on HSV-tk / GCV suicide gene therapy system killing of HCC CBRH7919 cells. The endogenous production of these lymphocytes Active substance may be Liu Wei Di Huang Wan on tumor suicide gene therapy efficacy of the material basis.