肝癌细胞系HepG2细胞周期与端粒酶活性及HBV复制的关系

Effect of Cell Cycle on Telomerase Activity of Hepatoma Cells and Its Relationship with Replication of Hepatitis B Virus

背景与目的:大量研究表明端粒酶活性与肿瘤细胞的发生、发展密切相关,而且乙型肝炎病毒(hepatitisBvirus,HBV)复制、端粒酶活性高低与细胞周期亦存在相关性。为进一步证实其内在联系,本实验探讨无血清培养、全反式维甲酸(all-trans-retinoicacid,RA)对HBVDNA转染的人肝癌细胞株HepG2细胞周期的影响以及细胞周期与端粒酶活性、HBV复制状态的关系。方法:分别用RA、无血清培养处理HepG2细胞并与对照组比较。细胞周期用流式细胞仪测定,端粒酶活性用TRAP-PCR-ELISA定量检测,HBVDNA分别用荧光PCR定量检测及斑点杂交半定量检测,HBsAg和HBeAg用ELISA方法定量检测。结果:RA、无血清培养使HepG2细胞生长受抑制,停滞在G0/G1期(RA组、无血清培养组G0/G1期时相百分比分别为68.3%、65.2%),端粒酶活性明显下降(RA组、无血清培养组代表端粒酶活性的吸光度A值分别为0.32、0.41),与对照组(G0/G1期时相百分比为43.1%,代表端粒酶活性吸光度A值为1.34)相比,两者之间有显著性差异(P<0.01);细胞培养液中HBVDNA、HBsAg和HBeAg含量显著增加(RA组分别为4.4×106copies/ml、3.5、19.8;无血清培养组分别为5.1×106copies/ml、3.7、22.5),与对照组(分别为1.2×106copies/ml、1.3、13.4)相比,两者之间有显著性差异(P<0.01)。结论:端粒?

BACKGROUND &OBJECTIVE:Previous studies have shown that the expression of telomerase activity is closely correlated with the formation and development of tumor cells. Furthermore, the cell cycle is associated with hepatitis B virus (HBV) replication level and telomerase activity. For further study their relationship, this experiment was designed to investigate the effect of serum deprivation or all trans retinoic acid(RA)on cell cycle of human hepatoma cells transfected by HBV DNA (HepG2 cell line) and the associations of cell cycle with telomerase activity and HBV replication. METHODS: Human hepatoma HepG2 cells were respectively treated with serum deprivation or RA. Cell cycle was analyzed using flow cytometry. Telomerase activity was determined quantitatively by TRAP PCR ELISA. HBV DNA in culture media was determined using quantitative PCR and semiquantitative dot blot hybridization assay. HBsAg and HBeAg in cell culture media were measured using quantitative ELISA. RESULTS: RA treatment or serum deprivation inhibited the proliferation of HepG2 cells and the cells were arrested at G0/G1 phase. The percentages of G0/G1 phase of RA group and serum deprivation were 68.3%and 65.2%, respectively, while that of control group was 43.1%(P< 0.01). The levels of telomerase activity also significantly decreased. The absorbance values that represented the telomerase activity of RA group and serum deprivation group were 0.32 and 0.41, respectively, while that of control group was 1.34(P< 0.01). In addition,HBV replication of HepG2 cells remarkably increased, which was shown as high products of HBV DNA, HBsAg and HBeAg in culture media of RA group and serum deprivation group. The contents of HBV DNAs were 4.4×106, 5.1×106, and 1.2×106 copies/ml in RA group, serum deprivation group, and control group, respectively(P< 0.01).The values of P/N of HBsAg were 3.5,3.7, and 1.3 in RA group, serum deprivation group, and control group, respectively(P< 0.01).The values of P/N of HBeAg were 19.8, 22.5, and 13.4 in RA group, serum deprivation group, and control group, respectively (P< 0.01). CONCLUSION: Telomerase expression was associated with cell cycle in HepG2 cells. Telomerase was mainly expressed in S phase of cell cycle. HBV replication was also closely correlated with cell cycle, which increased in quiescent hepatocytes and decreased in proliferating hepatocytes.