中文摘要：

目的探讨HBx与p16基因甲基化的关系,研究脱氢表雄酮（DHEA）对p16基因甲基化以及细胞周期和细胞凋亡的调节作用。方法以HepG2、HepG2/GFP和HepG2/GFP-HBx三种细胞为材料,采用MTT法检测细胞生长;流式细胞术检测细胞周期和凋亡率;MSP-PCR检测p16基因甲基化水平。比较分析HBx与p16基因启动子甲基化、DHEA与各检测指标的关系。结果HepG2/GFP-HBx细胞与HepG2细胞和HepG2/GFP细胞相比,细胞的增殖速度提高（P〈0.05）,G0/G1期细胞减少,S期细胞增多（P〈0.05）,凋亡率降低（P〈0.05）;HepG2/GFP-HBx细胞的p16基因启动子呈现高水平甲基化,HepG2和HepG2/GFP细胞呈现高水平非甲基化。100μmol/L的DHEA使三种细胞的增殖速度降低（P〈0.05）,G0/G1期细胞增加,S期细胞减少（P〈0.05）,凋亡率提高（P〈0.05）。DHEA可下调HepG2/GFP-HBx细胞的p16基因启动子甲基化水平,但对HepG2和HepG2/GFP细胞的p16基因启动子甲基化水平不产生影响。结论HBx引起肝癌细胞p16基因甲基化,并缩短细胞周期抑制细胞凋亡;DHEA可明显下调HBx引起的p16基因甲基化水平,延长细胞周期促进细胞凋亡,在无HBx基因整合的情况下,DHEA对肝癌细胞生长、细胞周期和凋亡的影响不通过p16基因甲基化的途径实现。

英文摘要：

Objective To explore the relationship of HBx and methylation of p16, and to study whether dehydroepiandrosterone （DHEA） regulates the methylation of p16, cell cycle and apoptosis. Methods The cell viability of HepG2, HepG2/GFP and HepG2/GFP-HBx cells was detected by MTT assay. The cell cycle and apoptosis rate was determined by flow cytometry. The methylation level of p16 was assayed by MSP-PCR methods. To compare and analyze the relationships of HBx and p16 promoter＇s methylation, DHEA to cell cycle and apoptosis. Results Compared with HepG2 and HepG2/GFP cells, the cell viability was increased （P 〈 0.05）, G0/G1 phases were decreased and S phase cells were increased （P 〈 0.05）, and the apoptosis rate was reduced （ P 〈 0.05） in HepG2/GFP-HBx cells. The p16 promoter showed a high methylation level in HepG2/ GFP-HBx cells, but high non-methylation level in HepG2 and HepG2/GFP cells. After treatment with 100 μmol/L DHEA in the three cell lines, the cell viabilities were reduced （ P 〈 0.05）, the number of G0/G1 ceils was increased （ P 〈 0.05）, that of S phase cells was reduced （ P 〈 0.05）, and apoptosis rate was increased （ P 〈 0.05）. DHEA down-regulated the methylation level of 1l6 promoter in HepG2/GFP-HBx cells, but no effect on HepG2 and HepG2/GFP cells. Conclusion HBx can induce methylation of p16 in liver cancer cells, shorten cell cycle and inhibit apoptosis. DHEA can down-regulate obviously methylation level of p16 which is induced by HBx, prolong cell cycle and promote apoptosis. On the contrary, in the case without HBx protein, the effects of DHEA on cell growth, cell cycle and apoptosis are carried out not by methylaion of p16.