脂蟾毒配基对人胃癌BGC-823细胞系细胞生长的干预效应  

Interventional effect of resibufogenin on the cell growth of human gastric cancer BGC-823 cell line

作　　者：孟书聪[1] 董晓敏[1] 肖军军[1] 王传社[2] 果德安[2] 

机构地区：[1]北京大学基础医学院细胞生物学系,北京市100083 [2]北京大学医学部中医药现代研究中心,北京市100083

出　　处：《中国临床康复》2006年第43期138-141,F0003,共5页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(330690);教育部教育振兴行动计划专项(985-2-016-24)~~

摘　　要：目的:观察中药蟾酥主要成分之一——脂蟾毒配基对体外培养人胃癌BGC-823细胞系细胞生长的干预及其与细胞色素C和半胱氨酸天冬氨酸蛋白酶3活化的关系。方法:实验于2004-09/2005-12在北京大学医学部细胞生物学系实验室完成。体外培养人胃癌BGC-823细胞系,脂蟾毒配基各组加入不同浓度的脂蟾毒配基(先用二甲基亚砜溶解,再经RPMI-1640培养液稀释至终浓度。细胞增殖及增殖抑制实验、细胞核形态观察及核DNA含量测定实验中脂蟾毒配基浓度分别为0.1,1,10μmol/L;细胞周期分布实验、细胞凋亡率实验、线粒体膜电位实验中脂蟾毒配基浓度分别为0,1,2.5,5μmol/L)。空白对照组加入RPMI-1640培养液。盐酸阿克拉霉素组加入5μmol/L盐酸阿克拉霉素。采用酸性磷酸酶法检测细胞增殖抑制作用;细胞荧光分光光度仪观察细胞核形态及核DNA含量测定;流式细胞仪检测细胞周期、细胞凋亡率及其线粒体膜电位变化;Westernblot检测与细胞凋亡相关基因蛋白表达的变化。结果:①经0.1,1,10μmol/L脂蟾毒配基分别作用24,48,72h后,细胞生长显著抑制,脂蟾毒配基对癌细胞的生长抑制百分率与剂量、时间呈正相关,其IC50分别为3.9,2.0,1.5μmol/L。②随脂蟾毒配基作用浓度和时间的延长,癌细胞核荧光强度减弱,细胞核DNA含量明显降低。③0.1,1μmol/L脂蟾毒配基作用24～48h后癌细胞阻滞在S期,5μmol/L脂蟾毒配基作用72h时,细胞阻滞在G2+M期;盐酸阿克拉霉素与癌细胞作用24～48h,细胞阻滞在S期,作用72h时细胞阻滞在G2+M期。④脂蟾毒配基作用后,细胞凋亡率明显高于对照组。⑤脂蟾毒配基引起细胞线粒体膜电位下降,释放入胞质中的细胞色素C增多,促进半胱氨酸天冬氨酸蛋白酶3蛋白活化,抑制Bcl-2蛋白表达,诱导细胞凋亡。结论:体外实验条件下,脂蟾毒配基抑制人胃癌BGC-823细胞生长并诱导细胞发生凋亡,其诱导凋亡�AIM： To observe the intervention of resibufogenin; one of main components of Chinese herb venenum bufonis, on the growth of human gastric cancer BGC-823 cell line cell cultured in vitro, and the relationship with cytochromc C and Caspase-3 activation. METHODS： The experiment was conducted in the Laboratory of Department of Cell Biology, Basic Medical College, Peking University from September 2004 to December 2005. BGC-823 cell line was cultured in vitro. Resibufogenin of different concentrations were added in each resibufogenin group （Firstly, dissolved with dimethyl sulphoxide, and then diluted with RPMI-1640 culture solution till final concentration; resibufogenin was 0.1, 1, 10 μmol/L in the cell proliferation and proliferation inhibition test, morphous observation of cell nucleus and DNA content determination tests; reslbufogenin was 0, 1, 2.5, 5μmol/L in the cell-cycle distribution test, cell apoptosis test, mitochondria membrane potential test）. RPMI-1640 cuhure solution was added in the blank control group. 5μmol/L aclarubicin hydrochloride was added in the aclarubicin hydrochloride group. Anti-proliferatian effect was measured with acid phosphatase assay （APA）. Cell morphological change and DNA content determination were observed with cell fluorescent spectrophotometer. Cell cycle, apoptotic rate and changes in mitochondrial transmembrane potential were measured with cytofluorometry. Change of cell apoptosis related gene protein was determined with Western blot. RESULTS： （1）After the BGC-823 cells were treated with 0.1, 1, 10 μmol/L resibufogenin for 24, 48 and 72 hours, the cell growth was inhibited significantly. The percentage of resibufogenin on grow inhibiting of cancer cell had positive correlation with dosage and time, and IC50 was 3.9, 2.0, 1.5 μmol/L, respectively.（2）With the prolongation of time and increase of concentration of resibufogenin, fluorescence intensity of cancer cell nucleus reduced and the content of cell nucleus DNA decreased obviously. （3）0.1, 1

关 键 词：蟾酥 细胞周期 线粒体 膜电位 细胞凋亡易感蛋白 

分 类 号：R735.2[医药卫生—肿瘤]