低相对分子质量肝素增强顺铂诱导肝癌细胞的凋亡作用

Potentiation of cisplatin induced apoptosis by low molecular weight heparin in human hepatocellular carcinoma cell

目的:探讨低相对分子质量肝素(low molecular weight heparin,LMWH)对顺铂(cisplatin,DDP)诱导肝癌细胞凋亡作用的影响及其作用机制。方法:实验分为对照组、LMWH组、DDP组、LWMH与DDP联合应用组。应用MMT法检测药物对肝癌SMMC-7721细胞增殖的影响;吖啶橙/溴化乙锭双荧光法和流式细胞术检测各组细胞凋亡情况;实时荧光定量PCR和Western印迹检测细胞凋亡相关蛋白Fas,Bcl-2和Bax的表达。结果:MTT检测结果显示:与对照组相比,DDP组及LWMH与DDP联合应用组的SMMC-7721细胞生长均有显著的抑制(均P<0.05);LMWH组的Fas,Bcl-2和Bax m RNA及蛋白均无明显的变化(均P>0.05);DDP组Fas表达量明显增加(P<0.05),而Bcl-2轻度降低(P<0.05),Bax无明显变化(P>0.05)。LWMH与DDP联合应用组与对照组及DDP组相比,Bcl-2表达明显降低(均P<0.05),Bax表达明显增高(均P<0.05);Fas表达量与对照组相比明显增加(P<0.05),但与DDP组相比增加不明显(P>0.05)。结论:LMWH具有增强DDP诱导肝癌SMMC-7721细胞凋亡的作用,其机制可能与LMWH增强DDP诱导的细胞凋亡中线粒体通路和促使细胞凋亡增加有关。

Objective:To explore the effects of low molecular weight heparin(LMWH) on cisplatin(DDP)-induced apoptosis in human hepatocellular carcinoma cell and the underlying mechanisms.Methods:Hepatocellular carcinoma SMMC-7721 cells were divided into a control group,a LMWH group,a DDP group and a LMWH plus DDP group.The effect of the drugs on proliferation of hepatocellular carcinoma SMMC-7721 cells were evaluated by 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di- phenytetrazoliumromide(MTT) assay,and the apoptosis were detected by acridine orange/ethidium bromide(AO/EB) double fluorescence method and flow cytometry method.The expression levels of apoptosis-related protein Fas,Bcl-2 and Bax were detected by quantitative realtime PCR and Western blot.Results:Compared with the control group,the proliferation of SMMC-7721 cells was inhibited in the DDP group and the LMWH plus DDP group(both P<0.05).The mRNA and protein levels of Fas,Bcl-2,Bax in the SMMC-7721 cells were not obviously changed in the LMWH group(all P>0.05).The Fas level was increased obviously(P<0.05),while the Bcl-2 level moderately reduced(P<0.05),Bax were not obviously changed in the DDP group(P>0.05).Compared with the control group and the DDP group,the Bcl-2 level was reduced significantly in the LMWH plus DDP group(both P<0.05),while the level of Bax was increased obviously(both P<0.05).Compared with control group,the Fas level was increased significantly in the LMWH plus DDP group(P<0.05),but the increase was not significant compared with the DDP group(P>0.05).Conclusion:LMWH can enhance the cisplatin-induced apoptosis in SMMC-7721 cells,which might be related to activation of mitochondrial pathway.