链球菌蛋白对人肝癌Bel-7402细胞的增殖抑制作用及其分子机制  

Inhibitory effects of streptococcal protein on the proliferation of human hepatocelluar carcinoma Bel-7402 cells: An investigation of the molecular mechanism

作　　者：李彦芹[1] 郑群[1] 王艳[1] 李卫红[2] 朱俊平[1] 平国玲[1] 范东瀛[1] 张力平[1] 

机构地区：[1]首都医科大学病原生物学系,北京市100069 [2]首都医科大学细胞生物学系,北京市100069

出　　处：《中国组织工程研究与临床康复》2007年第43期8733-8736,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：北京市教育委员会科技发展计划项目(KM200410025008)~~

摘　　要：目的:从分子水平观察链球菌蛋白质对人肝癌Bel-7402细胞的生长增殖的抑制作用。方法:实验于2005-10/2006-11在首都医科大学病原生物学系实验室完成。①链球菌32080株购自中国医学细菌保藏管理中心,人肝癌Bel-7402细胞株由首都医科大学细胞生物学系惠赠。②常规培养链球菌并按照Winters方法提取细菌蛋白。③用0,10,20,100mg/L细菌蛋白作用于单层培养的Bel-7402细胞24,48,72h,采用MTT法检测链球菌蛋白对细胞的增殖抑制作用。④Bel-7402细胞经10mg/L或50mg/L细菌蛋白分别作用48,72h后,流式细胞术检测细菌蛋白对癌细胞生长周期时相分布。⑤Bel-7402细胞经50mg/L的细菌蛋白分别作用24,48,72h后,Annexin-v PI双染法检测Bel-7402细胞凋亡率。⑥用50mg/L的链球菌蛋白分别作用于Bel-7402细胞0,24,48,72h后,Western Blot检测链球菌蛋白对Bel-7402细胞凋亡相关蛋白的影响。结果:①链球菌蛋白对Bel-7402细胞的抑制作用:链球菌蛋白能显著抑制人肝癌Bel-7402细胞增殖,100mg/L细菌蛋白作用于细胞24,48,72h的抑制率分别为15.6%,35.6%,50%,呈剂量和时间依赖关系。②细菌蛋白对肝癌细胞生长周期时相分布及细胞凋亡率的影响:经链球菌蛋白(10,50mg/L)作用后,G2/M期Bel-7402细胞比率较正常对照组增加,差异有显著性(P<0.05);流式细胞术检测显示,经50mg/L链球菌蛋白作用后,实验组Bel-7402细胞凋亡率明显高于对照组细胞(P<0.01)。③Bel-7402细胞凋亡相关蛋白的变化:Bel-7402细胞经50mg/L链球菌蛋白分别作用24,48,72h后,细胞色素C的释放逐渐增多,Procaspase-3及Bcl-2蛋白水平降低。结论:链球菌32080株蛋白对人肝癌Bel-7402细胞的生长抑制作用存在着剂量和时间依赖性,并能将细胞的生长周期阻滞于G2/M期,诱导细胞凋亡,影响Bcl-2,procaspase-3和细胞色素C等重要的凋亡相关蛋白水平。AIM： To observe the inhibitory effects of streptococcal protein on proliferation of human hepatocelluar carcinoma Bel-7402 cells from molecular level. METHODS： The experiment was conducted in the Department of Pathogenic Biology of Capital Medical University from October 2005 to November 2006. （1）Streptococcus （No. 32080） was bought from the National Center for Medical Culture Collection. Human hepatocelluar carcinoma Bel-7402 cells were presented as a gift from the Department of Cytobiology of Capital Medical University. （2）Streptococcal protein was extracted by Winters method after cultivating streptococcus conventionally. （3）Bel-7402 cells were treated with different concentrations （0, 10, 20, 100 mg/L） of streptococcal protein for 24, 48 and 72 hours, and then MTT assay was used to evaluate the inhibitory effect of it. （4）Bel-7402 cells were treated with 10 mg/L or 50 mg/L streptococcal protein for 48 and 72 hours, respectively. Flow cytometry was applied to measure the distribution of growth cycle phase of malignant cells. （5）After treating with 50 mg/L streptococcal protein for 24, 48 and 72 hours, respectively, the apoptotic rate of Bel-7402 cells was measured by Annexin-v PI double-staining. （6） After treating with 50 mg/L streptococcal protein for 0, 24, 48 and 72 hours, respectively, the effect of streptococcal protein on apoptotic related protein of Bel-7402 cells was measured by Western Blot. RESULTS： （1）lnhibitory effects of streptococcal protein on Bel-7402 cells： Streptococcal protein inhibited the proliferation of Bel-7402 cells significantly with time-dependent and dose-dependent effects, and the inhibitory rates were 15.6%, 35.6% and 50% with 100 mg/L streptococcal protein for 24, 48 and 72 hours, respectively. （2）Effects of streptococcal protein on cell cycle and apoptotic rate of Bel-7402 cells： Bel-7402 cells treated with the protein （10, 50 mg/L） were increased in GJM phase as compared with the normal control group, and there were signifi

关 键 词：链球菌 细胞凋亡 人肝癌Bel-7402细胞 流式细胞术 

分 类 号：R735.7[医药卫生—肿瘤]