【摘要】 目的从蛤蚧组织液中筛选对肝癌HepG-2细胞具有生长抑制作用的蛋白组分,体外研究其抗肿瘤作用及机制。方法提取蛤蚧蛋白,用sephadexG-50凝胶层析分离出不同分子量的蛤蚧蛋白组分,采用MTS法观察蛤蚧蛋白各组分对肝癌HepG-2细胞的生长抑制作用;Hoechst33342/PI双荧光染色法观察其作用前后的细胞凋亡,并用RT-PCR法检测蛤蚧蛋白组分作用后bax、bcl-2、cmyc和p53基因的表达情况。结果 sephadexG-50凝胶层析可分离出3种分子量不同的蛤蚧蛋白组分,3种蛤蚧蛋白组分都具有不同程度地抑制HepG-2细胞生长的作用,其中分子量为3～20KD的蛤蚧蛋白组分抑制作用最强,且具有剂量依赖性。Hoechst33342/PI双荧光染色法检测显示3～20KD蛤蚧蛋白作用后坏死细胞和凋亡细胞均增加,RT-PCR法检测发现3～20KD蛤蚧蛋白组分作用后c-myc基因mRNA表达水平稍降低,p53基因mRNA表达水平升高,但两者差异均无统计学意义,bax基因mRNA表达水平明显增高,差异有统计学意义,bcl-2基因在蛋白作用前后均无表达。结论实验提取的蛤蚧蛋白对肝癌HepG-2细胞具有生长抑制作用,分子量3～2KD的蛤蚧蛋白组分抑制作用最强。其作用机制可能为蛤蚧蛋白通过调高肿瘤bax基因mRNA表达水平诱导肝癌HepG-2细胞凋亡。更多还原

【Abstract】 ObjectiveTo investigate the suppressive effect of gecko protein on growth of liver cancer cell HepG-2.Methods Three gecko protein components with different molecular weights were isolated and purified through G-50 Sephacryl chromatograph.MTS method was used to observe the growth inhibiting rate (GIR) of each gecko protein components.Immunofluorescence microscopy (Hoechst 33342/PI) was used to observe apoptosis.RT-PCR assay was used to detect the expression of gene bax,bcl-2,c-myc and p53.Results All of the three gecko protein components showed suppressive effect on growth of HepG-2 cells,while the component 3～20KD was not only more effective than the other two but also its efficiency showed dose-dependent.The results of Hoechst 33342/PI showed that exposure of HepG-2 cells to 3～20KD gecko protein resulted in increased necrosis and apoptosis,increased expression of p53 and bax as well as decreased expression of c-myc.Conclusions 3~20KD gecko protein possesses the ability of inhibiting growth of HepG-2 cells.The mechanisms of which may involve the enhancement of bax gene expression.更多还原