目的探讨没食子酸(gallic acid,GA)抑制人肝癌细胞SMMC-7721增殖的作用,揭示其促凋亡的相关分子机制。方法体外培养人肝癌细胞SMMC-7721,MTT法观察细胞在GA作用24、48、72h后的增殖情况;用倒置显微镜观察细胞形态学的变化;透视电镜观察细胞内部结构变化;AnnexinV-FITC/PI检测细胞凋亡;采用RT-PCR技术研究p53mRNA的变化;应用Westernblot法检测p53蛋白水平的表达。探讨GA对人肝癌细胞SMMC-7721的诱导凋亡作用及机制。结果剂量为6.25—50umol·L-1 GA作用SMMC-7721细胞48h有明显的增殖抑制活性,引起核固缩、凝聚、碎裂,诱导细胞凋亡,并呈剂量依赖性;RT-PCR和Western blot结果显示,GA能升高p53mRNA水平和p53蛋白表达。结论GA可抑制人肝癌细胞SMMC-7721的增殖,诱导凋亡,可能与其上调肿瘤相关抑癌基因p53有关。
Aim To investigate the proliferative effect and the apoptosis of human hepatoma SMMC-7721 ceils induced by gallic acid (GA), and its underlying mechanism. Methods SMMC-7721 cells were cultured in vitro. MTT assay was used to observe the pro- liferation of SMMC-7721 cells induced on GA 24, 48, 72 h. The morphological and ultra structural changes of the SMMC-7721 cells were observed by inverted microscope and transmission electron microscope respective- ly. Annexin V-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population. The expression of p53 mRNA was investigated by RT-PCR. Western blot was used to determine the protein expression of p53. Results GA(6.25 - 50 umol . L-1) markedly inhibited the activity of proliferation and induced apoptosis of SMMC-7721 cells after 48h in a dose-dependent manner. GA significantly induced cell nuclear condensation and fragmentation. RT-PCR and Western blot results showed that GA could improve the expression of p53 mRNA and protein. Conclusion GA can inhibit the proliferation of human hepatoma SMMC-7721 ceils and induce cells apoptosis. The mechanism may be associated with improving tumor suppressor gene p53 expression.