补益与解毒化瘀方协同DDP对SKOV3细胞增殖与凋亡的影响

Comparative Study of that Synergistic Effect of Cisplatin Combined with Compound Herbal Medicinal Prescription for Tonic Quality and Activating Blood Circulation is on SKOV3 Cell Proliferation and Apoptosis

目的观察抗癌中药复方(CHM P)药血清及其协同顺铂(DDP)对卵巢癌细胞SKOV 3的增殖及凋亡的影响,分析补益(CHM P1)与解毒化瘀(CHM P2)复方在体外对顺铂协同作用的最佳搭配方式及其机制。方法制备复方CHM P1和CHM P2颗粒药不同时相、不同剂量的药血清;M TT法测定各时相及不同剂量药血清对SKOV 3细胞增殖的影响,确定药血清最佳时相及剂量,测定两药物相互作用指数(coeffic ien t of drug in teraction,CD I),观察CHM P与DDP之间的协同作用;流式细胞术(FCM)分析对照组(A组)、CHM P1组(B组)、CHM P2组(C组)、DDP组(D组)、CHM P1+DDP(E组)、CHM P2+DDP组(F组)等各组药血清对SKOV 3细胞凋亡的影响,DNA琼脂糖凝胶电泳法观察上述各实验组细胞凋亡,W estern b lot检测上述各组细胞TNFR 1、C aspase-8蛋白表达。结果2t-1时相、中剂量CHM P1、CHM P2血清及大剂量DDP血清对SKOV 3细胞的抑制率最好,CHM P1、CHM P2与DDP两两药物之间具有明显的协同作用,CD I分别为0.66、0.58;F组对SKOV 3细胞的抑制率优于E组(P<0.05)。各实验组血清作用SKOV 3细胞48 h后,琼脂糖电泳显示出典型的凋亡梯度状条带;联合用药组对SKOV 3细胞凋亡的影响强于单独用药组(P<0.05)。W estern b lot分析显示联合应用后TNFR 1、C aspase-8蛋白表达增多,与联合应用组细胞凋亡率增加相一致。结论CHM P药血清抑制人卵巢癌细胞SKOV 3生长的作用主要是通过诱导细胞凋亡实现,与化疗药DDP具有协同作用,解毒化瘀复方与DDP的协同作用优于补益复方。CHM P与DDP协同作用与其促进凋亡途径TNFR 1启动及C aspase-8的活化有关。

Obiective To investigate the synergistic effects on cell apoptosis and growing restriction of SKOV3 cells by the combination of compound herbal medicinal prescription （CHMP） with cisplatin （DDP）. Methods Cisplatin and two CHMP for tonic quality（CHMP1） and activating blood circulation （CHMP2）, which was medicated serum, were prepared and used to treat the human ovarian carcinoma cell line SKOV3. By serum pharmacologic method, the growth and apoptosis of SKOV3 cell were observed at different time points （24, 48, 72, 96 h） with different concentrations of medicated serum. Coefficient of drug interaction （CDI） between CHMP1 and CHMP2 was studied by MTT method. The effects of control group （A group）, CHMP1 group （B group）, CHMP2 group （C group）, DDP group （D group）, CHMP1＋DDP group （E group）, CHMP2＋DDP group（Fgroup） to SKOV3 cell were studied by flow cytometry; and the cell apoptosis was observed by agarose electrophoresis; the expressions of TNFR1, caspase-8 on each group were analyzed by Western blot method. Results Synergistic effects were found between herbal medicinal mixtures and DDP, Restraining rate of SKOV3 and CHMP serum concentration was not in a dose-dependent manner as DDP was. CDI between CHMP1 and CHMP2 was found to be significant difference （CDI of CHMP1, CHMP2 and DDPwas 0.66, 0.58 respectively）. It showed that the combined treatment was able to get better effect than single drug treatment. The performed agarose electrophoresis revealed the extracted DNA to show a typical ladder patterns for cell apoptosis. The analysis results of western blot showed the increased expressions of TNFR1 and caspase-8 after combined using of medicine, which were accord to the rates of apoptosis. Conclusions CHMP drug granules show the synergistic effects with DDP, and the suppressing functions in the course of cell proliferation, and the inducing effect on apoptosis of human ovarian carcinoma cell line SKOV3 in vitro. And this mechanism is showed to be sponsored by the activation of TNFR1 and Caspase-8.