黄芩素对肝癌HepG2细胞增殖和凋亡的影响及miR34a在其机制中的作用.doc

黄芩素对肝癌HepG2细胞增殖和凋亡的影响及miR-34a在其机制中的作用 　　作者：宋超1,刘霞2,邵启祥2,徐三荣1　　作者单位：1.江苏大学附属医院普外科， 江苏 镇江 212001; 2.江苏大学基础医学与医学技术学院， 江苏 镇江 212013 　　【摘要】目的：观察12-脂氧合酶抑制剂黄芩素(Baicalein)对人肝癌细胞株HepG2增殖和凋亡的影响，并初步研究miR-34a在黄芩素影响HepG2细胞机制中的作用。方法：黄芩素(浓度分别为25、50和100 Μmol/L)作用HepG2细胞24 h和48 h后，分别用Hoechst33342染色后荧光显微镜观察HepG2细胞形态变化，CCK8法检测细胞增殖活性，流式细胞术检测细胞凋亡的情况。采用qRT-PCR检测HepG2细胞中miR-34a的表达。结果：黄芩素(浓度分别为25、50、100 Μmol/L)作用24、48 h后，HepG2细胞数量明显减少，细胞核固缩或裂解;CCK8法显示细胞增殖受到抑制;流式细胞术检测显示DNA染色荧光强度下降，黄芩素作用24 h后细胞出现DNA亚二倍体峰的比率分别为16.10%、33.18%和68.34%,48 h后为19.55%、36.59%和74.08%，活细胞比率下降，细胞死亡率增加。黄芩素处理后的HepG2细胞中miR-34a的表达量较未处理组明显增高。结论：黄芩素能抑制HepG2细胞的增殖，促进其凋亡，其机制可能与miR-34a上调有关。 　　【关键词】 脂氧合酶抑制剂,黄芩素,HepG2细胞; 细胞凋亡; miR-34a 　　[Abstract]　Objective： To investigate the effects of 12-lipoxygenase inhibitor Baicalein on human hepatocellular carcinoma cell line HepG2， and the role of miR-34a in the mechanism of Baicalein effect on HepG2 cells. Methods： HepG2 cells were treated by Baicalein within different concentrations (25， 50， 100 Μmol/L ) 24， 48 h respectively. The morphological changes of HepG2 cells were observed under fluorescence microscope after stained with Hoechst33342. Cell proliferation was validated by CCK8. Flow cytometry (FCM) was used to analyse the DNA content of HepG2 cells stained with PI. miR-34a of HepG2 cells was detect by qRT-PCR after treated with or without Baicalein. Results： Obvious appearances of cell apoptosis were observed under fluorescence microscope stained with Hoechst 33342， including chromatin condensation， nuclear fragmentation， and apoptotic bodys forming. The result of CCK8 assay showed that the cell proliferation of HepG2 was inhibited in a dose and time dependent manner. A decrease of fluorescence intensity of DNA of HepG2 cells with PI staining was demonstrated by FCM and accompanied with the presence of the hypodiploid peak. The percentage of cells with hypodiploid DNA content of HepG2 cells， treated with three different concentrations of Baicalein for 24 h， was 16.10%，