丹参注射液和三七总皂甙对大鼠肝癌细胞及自杀基因旁观者效应的影响

Effects of Danshen and Total Saponins of Panax Notoginsen on rat hepatocarcinoma cells and bystander effect of suicide gene

目的:探讨丹参注射液和三七总皂甙对大鼠肝癌细胞及自杀基因旁观者效应影响.方法:丹参注射液和三七总皂甙以不同浓度与GCV分别或共同作用于大鼠肝癌CBRH7919的tk-细胞,以及含5-10%tk+细胞的tk+和tk-混合细胞,MTT检测各组存活率(n=3,6),两两比较分析各组存活率的差异,并用q值分析中药与自杀基因系统联合的相互作用是否具有协同性.q值为联合用药时实测药效与理论药效的比值,q≤0.85为拮抗作用,0.85≤q<1.15为相加作用,q≥1.15为协同作用.结果:5,10,20,40mL/L的丹参注射液作用于CBRH7919细胞,72h存活率分别为81.0±17.3%,55.6±12.0%,14.6±4.4%,11.5±0.9%;IC50为11.4mL/L.丹参注射液联合tk/GCV系统对肝癌细胞的作用:5%tk+/GCV组存活率为78.9±27.9%,GCV组为84.3±18.2%,前者比后者仅多了5%tk+细胞,相对抑制率7.6%;5mL/L丹参联合5%tk+/GCV组存活率为47.8±14.5%(q=1.60),5mL/L丹参+GCV组的存活率为72.8±4.5%,前者比后者唯一差异条件也是多了5%tk+细胞,相对抑制率34.3%(P=0.049);10mL/L丹参联合5%tk+/GCV组的存活率为12.2±5.9%(q=1.46),10mL/L丹参+GCV组的存活率为36.5±2.7%,前者比后者仅多了5%tk+细胞,相对抑制率66.6%(P=0.003).10,50,100,140mg/L三七总皂甙作用72h细胞存活率分别为104.1±3.7%,107.6±3.1%,69.7±8.5%,59.3±2.9%;IC50为220mg/L.三七总皂甙联合tk/GCV系统对肝癌细胞的作用:10%tk+/GCV组比GCV组存活率下降了17.2%(P<0.05),50mg/L和100mg/L三七总皂甙联合10%tk+/GCV组存活率(q=0.89,0.87)分别比相应的三七总皂甙+GCV组下降了17.7%和18.3%.结论:丹参注射液有明显抑制癌细胞生长作用,并能协同性增强tk/GCV系统对癌细胞的杀伤作用,增强自杀基因旁观者效应.三七总皂甙能一定程度抑制癌细胞生长,但对tk/GCV系统杀伤癌细胞只有加和作用,未发现有协同性作用.

AIM： To explore the effects of Danshen and Total Saponins of Panax Notoginsen （PNS） on rat hepatocarcinoma cells and the bystander effect of herpes simplex virus thymidine kinase gene/ganciclovir （HSV-tk/ GCV）. METHODS： Rat hepatocarcinoma cell line CBRH7919 （tk^-）, CBRH7919 （tk^±） and the 5-10% tk^± mixed cells were treated with diverse concentrations of Danshen injection, PNS, GCV separately, or Danshen injection plus GCV and PNS plus GCV （/7 = 3, 6）. The survival rate of the cells in each group was examined by MTT method, and q-value analysis was used to estimate the synergistic effect of traditional Chinese medicine on the suicide gene system. The q-value was equal to the ratio of the actual effect of combined treatment to its theoretical effect. The effect was classified into three categories： antagonistic effect （q ≤0.85）, additive effect （0.85 ≤ q 〈1.15）, and synergistic effect （q 〉1.15）. RESULTS： After the treatment of 5, 10, 20, 40 mL/L Danshen injection for 72 h, the survival rate of CBRH 7919 （tk^-） was 81.0±17.3%, 55.6±12.0%, 14.6±4.4%, and 11.5±0.9%, respectively. IC50 was 11.4 mL/L. The survival rate of the hepatocarcinoma cells was 78.9± 27.9% in the 5% tk^IGCV group, and 84.3±18.2% in the GCV group, and the survival rate in the former was 7.6% lower than that in the latter. The survival rate of the cells was 47.8±16.5% （q = 1.60） in the group of 5 mL/L Danshen injection combined with 5% tk^1± GCV, and 72.8±4.5% in the group of 5 mL/L Danshen injection combined with GCV, and the survival rate in the former was 34.3% lower than that in the latter. The survival rate was 12.2±5.9% （q = 1.46） in the group of 10 mL/L Danshen injection combined with 5% tk^±1 GCV, and 36.5±2.7% in the group of 10 mL/L Danshen injection combined with GCV, and the survival rate in the former was 66.3% lower than that in the latter. The survival rate of CBRH7919 （tk^-） was 104.1±3.7%, 107.6 ±3.1%, 69.7±8.5%, and 59.3±2.9%, respectively, after the treatment of 10, 50, 100, and 140 mg/L PNS for 72 h. ICs0 was 220 mg/L. The survival rate in the 10% tk^±IGCV group was 17.2% （P 〈0.05） lower than that in the GCV group. In the group of 50 mg/L and 100 mg/L PNS combined with 10% tk^±1GCV （q = 0.89, 0.87）, the survival rates were 17.7% and 18.3% lower than those in the corresponding groups of PNS combined with GCV, respectively. CONCLUSION： Danshen injection can inhibit the proliferation of hepatocarcinoma cells significantly, and can synergically enhance the killing and bystander effect of tk/GCV system. PNS can also inhibit the proliferation of hepatocarcinoma cells to some extent, but it has only additive effect on the tk/GCV system, while no synergetic effect.