外源性层黏连蛋白在姜黄素对人肝癌HepG_2细胞生长抑制作用中的影响  

Influence of laminin in cell growth inhibition of curcumin on human hepatocellular carcinoma HepG_2 cells

作　　者：霰海萍[1] 孟书聪[1] 董晓敏[1] 张莎[1] 尚应辉[1] 肖军军[1] 

机构地区：[1]北京大学基础医学院细胞生物学系,北京100191

出　　处：《解剖学报》2011年第1期38-44,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资金资助项目(30330690);教育部教育振兴行动计划专项资助项目(985BMU-2010-0063)

摘　　要：目的探讨在外源性层黏连蛋白(LN)与其受体结合下,姜黄素对人肝癌HepG2细胞生长、凋亡的影响。方法实验分为对照组、LN组(20μg/L LN)、姜黄素组(40μmol/L姜黄素)、联合组(20μg/L LN+40μmol/L姜黄素)。采用酸性磷酸酶法(APA)、流式细胞术(FCM)和Western blotting法等探讨在外源性LN与其受体结合下,姜黄素对人肝癌HepG2细胞生长、细胞凋亡率、线粒体膜电位、细胞增殖相关蛋白α-蛋白激酶(α-PKC)及细胞凋亡相关蛋白人多聚ADP核糖聚合酶(PARP)、Caspase-3、Bcl-2和p53表达的影响。结果 LN组与对照组相比,细胞存活率增加。姜黄素组与联合组能显著抑制人肝癌HepG2细胞的增殖,并呈时间依赖性。姜黄素组与联合组作用48h时,倒置显微镜下,细胞数量明显减少,皱缩变圆,大部分细胞悬浮;中晚期凋亡和坏死细胞比率(%)分别为97.04±1.50,98.02±1.35;细胞内钙离子浓度升高;线粒体膜电位下降;增殖相关蛋白α-PKC含量减少;凋亡相关蛋白PARP出现剪切带,Caspase-3表达下调,p53表达上调,Bcl-2表达无明显变化。结论在外源性层黏连蛋白与其受体结合下,姜黄素与人肝癌HepG2细胞作用后抑制生长并诱导细胞发生凋亡;显示出姜黄素抗肿瘤作用稳定,其机制可能与上调p53,下调α-PKC有关。Objective To study the influence of curcumin on cell growth and apoptosis in HepG2 cell, in the presence of exogenous laminin（LN） to its receptor. Methods HepG2 cells were cultured in DMEM containing 10% fetal bovine serum at 37℃ in a humidified atmosphere with 5% CO2. The experiment was performed in four groups： the control group, LN group （20μg/L LN） , eurcumin group （40p, mol/L cureumin） , and combination group （ 20μg/L LN ＋ 40μmol/L eurcumin ）. Acid phosphatase assay （APA）, flow eytometry （FCM） and Western blotting were used to detect the cell viability, apoptosis ratio, intracellular calcium concentration , mitochondrial transmembrane potential, expression of proliferation-related proteinα-PKC and apoptosis-related protein poly ADP ribose polymerase（PARP） , Caspase-3, p53 and Bcl-2. Results Compared with the control group , LN group increased the viability rate. Curcumin （40μmol/L） and combination（ 20μg/L LN ＋ 40μmol/L curcumin ） inhibited HepG2 cells proliferation obviously, somewhat in a time dependent manner. After treatment with 40μmol/L cureumin and 20μ g/L LN ＋ 40μmol/L eurcumin for 48hours, the number of HepG2 cells reduced, volums shrank, shape rounded and suspended mostly. The rates of late apoptosis and necrosis cells（ % ） were 97.04 ±1.50,98.02 ±1.35. Intraeellular calcium concentration rised. Mean fluorescence intensity （MFI） of mitochondrial transmembrane potentials in HepG2 cells dropped. The expression of proliferation-related proteinα- PKC decreased. Curcumin could induce cleavage of PARP. The expression of apoptosis-related protein p53 increased, Caspase-3 decreased but Bel-2 had no obvious change. Conclusion Curcumin inhibited cell proliferation and induced apoptosis of HepG2 cell,which was not influenced under exogenous laminin to its receptor and showed well stability of antitumor. The mechanism might relate to down-regulating the expression of α-PKC, and up- regulating expressionof p53.

关 键 词：层黏连蛋白 姜黄素 HepG:细胞 免疫印迹法 流式细胞术 

分 类 号：R730.52[医药卫生—肿瘤]