蟾毒灵诱导人宫颈癌HeLa细胞凋亡的蛋白组学研究

Study on proteomics of HeLa cell apoptosis in bufalin-induced human cervical carcinoma

目的:该文通过考察蟾毒灵(bufalin)处理人宫颈癌HeLa细胞株后对细胞蛋白质表达谱的影响,寻找蟾毒灵在HeLa细胞中的可能作用靶点。方法:通过MTT检测细胞活性确定蟾毒灵的IC50,并通过流式细胞术和Hoechst 33342染色检测蟾毒灵诱导细胞凋亡。应用蛋白质组学技术寻找差异表达蛋白点并进行鉴定,用Western blotting试验进行确认。结果:蟾毒灵具有较强的细胞毒作用,IC50(154±21.5)nmol.L-1,可以诱导HeLa细胞发生凋亡。蛋白组学的结果显示蟾毒灵处理组与对照组相比有11个差异表达蛋白,其中9个蛋白点发生了下调;2个蛋白发生了上调。这些蛋白可能是蟾毒灵在HeLa细胞中的作用靶点。Western blotting分析显示heat shock protein 27,vimentin,HNRPK在蟾毒灵处理后的表达调节结果与双向电泳的结果吻合。结论:蟾毒灵可以诱导人宫颈癌HeLa细胞发生凋亡,其凋亡诱导作用可能是多个靶点蛋白共同参与的结果。

Objective： To seek possible effect targets of bufalin in HeLa cells by studying the impact of bufalin on cell protein expression profile after treatment on human cervical carcinoma cell lines HeLa. Method： Bufalin＇s IC50 was measured by MTT assay. The apoptosis of cells was observed by FCM （flow cytometry） and Hoechst 33342 staining assay. Differentiated expression protein spots were founded and identified using proteomic techniques, which could induce HeLa cell apoptosis. Result： Bufalin showed remarkable cytotoxic effect on HeLa cells. IC50 （154 ±21.5 ） nmol .L^- 1 indicated the possibility of inducing cell apoptosis. The protein expression profile showed 11 differentiated expression protein spots. Among the 11 proteins, nudix-type motif 5, vimentin, hnRNP C1/hnRNP C2 variant, HNRPK, HNRPK isoform a variant （two spots are the same protein） , heat shock protein 27, macrophage-capping protein, SELENBP1 protein were down-regulated, while ribosomal protein, large, PO and S-adenosylmethionine synthetase 2 were up-regulated by bufalin treatment. They may be effect targets of bufalin in HeLa cells. Western blotting showed consistent results in heat shock pro- tein 27, vimentin and HNRPK between expression after treatment with bufalin and two-dimensional electrophoresis. Conchlsion ： Bufa- 1in can induce apoptosis in human cervical carcinoma cells HeLa and the effect of bufalin may be related to the joint intervention with multiple protein targets.