葡萄籽原花青素诱导人肝癌HepG2细胞凋亡及自噬性死亡  

Grape seed proanthocyanidins induce apoptosis and autophagic cell death in human hepatoma HepG2 cells

作　　者：王威[1] 陈景红[1] 王新宁[2] 李强[1] 陈庆[1] 蒋建伟[1] 

机构地区：[1]暨南大学医学院生化教研室,广东广州510632 [2]广东药学院附属第一医院肿瘤科,广东广州510080

出　　处：《暨南大学学报（自然科学与医学版）》2011年第2期181-187,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省科技计划项目(2009B080701051);广东省医学科研基金项目(A2009348)

摘　　要：目的:探讨原花青素对肝癌HepG2细胞的抑制作用及其可能的作用机制。方法:改良MTT法(WST-8法)及克隆形成抑制实验观察原花青素对肝癌HepG2细胞的生长抑制作用,碘化丙锭(PI)单染色检测细胞周期改变,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平,激光共聚焦显微镜观察和Western blotting检测细胞自噬发生。结果:原花青素作用HepG2细胞后,不同质量浓度的原花青素对肝癌细胞的生长均有抑制作用,且呈剂量-效应关系(P<0.01),原花青素作用于人肝癌HepG2细胞48 h的IC50为1.304×10-1g/L;与对照组相比随着原花青素作用于HepG2细胞的浓度增加,细胞克隆逐渐减少;随着原花青素浓度的增加,人肝癌HepG2细胞出现明显G1期阻滞,且高浓度原花青素组出现明显的亚二倍体峰,当原花青质量素浓度达到1.6×10-1g/L时,亚二倍体百分率为81%;不同质量浓度原花青素作用后,出现明显的凋亡细胞及坏死细胞、自噬性死亡和非特异性死亡细胞群;经原花青素处理转染GFP-LC3质粒的HepG2细胞胞浆内,LC3呈现明显的点状聚集;细胞自噬标志蛋白LC3-II的蛋白表达水平随着原花青素浓度增加逐渐增多。结论:原花青素通过诱导细胞凋亡及自噬性死亡的方式抑制人肝癌细胞的生长。Aim：To investigate the inhibiory effect of procyanidins on human hepatoma HepG2 cell lines and the possible mechanism.Methods： Improved MTT assay（WST-8 method） and cell colony formation inhibitory assay were used to investigate the inhibition effect of procyanidins on human hepatoma HepG2 cell lines.Changes in cell cycle,DNA ploidy and apoptotic cell percentage were calculated by flow cytometry.Laser confocal microscopy and Western blotting assay were used to determine cell autophagy.Results： The results of WST-8 method shows that procyanidins with different concentrations inhibited human hepatoma HepG2 cells in a dose-dependent manner.The IC50 for procyanidins against human hepatoma HepG2 cells for 48 hours was 1.304×10-1g/L.Cell colone formation inhibition assay demonstrated that cell clones were decreased with increase of the concentration of procyanidins.PI staining analysis showed that cell cycle was arrested in G1 phase.DNA ploidy analysis showed that the percentage of hypodiploid cells was accumulated in a dose-dependent manner.Annexin V-FITC/PI staining analysis also showed a dose-dependent effect on late apoptotic and necrotic cells.By using laser scanning confocal microscopy it was observed that LC3 aggregates significantly,the green fluorescence of control group was dispersed.Western-blotting assay showed that the LC3-II protein expression levels increased gradually with increasing of procyanidins.Conclusion：Procyanidins inhibits the cell survival of human hepatoma HepG2 cells in a dose-dependent manner and arrestes cells at G1 phase.Procyanidins inhibits cell growth by inducing apoptotic and autophagic death.

关 键 词：原花青素 人肝癌HEPG2细胞 凋亡 自噬 

分 类 号：R285.5[医药卫生—中药学]