乙型肝炎病毒MHBs^(t155)蛋白促进肝癌细胞HepG2生长的实验研究

Hepatitis B virus MHBs^(t155) protein promotes growth of HepG2 cells

目的研究乙型肝炎病毒羧基末端155位截短的中表面蛋白(MHBst155)对肝癌细胞生长、增殖的影响。方法以Hep G2细胞和稳定表达GFP/MHBst155融合蛋白的Hep G2/GFP-MHBst155作为实验细胞,细胞用5-氮-2′-脱氧胞苷(5-Aza-Cd R,20μmol/L浓度)处理。采用四甲基偶氮噻唑蓝(MTT)法检测细胞吸光度值A490,分析细胞生长增殖情况;流式细胞术检测细胞周期变化。结果 72 h时Hep G2细胞组和Hep G2/GFP细胞组的A490分别为(0.67±0.12)、(0.70±0.06),与Hep G2/GFP-MHBst155细胞组(1.82±0.09)比较,差异均有统计学意义(t=-27.05、-36.71,P值均<0.05),而Hep G2细胞组与Hep G2/GFP细胞组比较差异无统计学意义(t=-1.21,P=0.24)。流式细胞术检测显示Hep G2/GFP-MHBst155细胞组的G0/G1期细胞比例为(26.23±2.70),明显低于Hep G2细胞组(45.20±1.25)和Hep G2/GFP细胞组(41.83±2.14),差异均有统计学意义(t=11.03、7.84,P值均<0.05);而经5-氮-2′-脱氧胞苷(5-Aza-Cd R)处理的Hep G2/GFP-MHBst155细胞组G0/G1期的比例为(43.03±1.45),与未处理Hep G2/GFP-MHBst155细胞组比较,差异有统计学意义(t=9.49,P<0.05)。结论 MHBst155可能通过缩短Hep G2细胞生长的G0/G1期而加快细胞周期进程,从而促进肝癌细胞的生长,其机制可能与MHBst155蛋白诱导生长调控基因的异常甲基化有关。

Objective To explore the effect of C-terminally truncated middle hepatitis B virus surface proteins（MHBs^t155） on cell cycle and growth of human hepatocellular carcinoma cells. Methods Hep G2 / GFP-MHBs^t155 cell line stably expressing the fusion protein of a green fluorescent protein（GFP） and MHBs^t155 was used in the experiment. Cell growth was evaluated by MTT method. Cell cycle was determined by flow cytometry in these cells with or without treatment of 5-aza-2′-deoxycytidine（5-Aza-Cd R, 20 μmol / L）. Results The A490 value in Hep G2 / GFP-MHBs^t155 was 1.82±0.09 at 72 hours of culture, and there was significant difference compared with Hep G2 cell（0.67 ±0.12, t =-27.05, P 〈0.05） and Hep G2 / GFP cell（0.70 ±0.06,t=-36.71, P0.05）. FCM analysis showed the percentage of G0/ G1 phase of Hep G2 / GFP-MHBs^t155（26.23±2.70）%, was significant lower compared with Hep G2（45.20±1.25, t=11.03, P0.05） and Hep G2 / GFP（41.83±2.14, t=7.84, P〈0.05）. Furthermore,in Hep G2 / GFP-MHBs^t155 cells with 5-Aza-Cd R treatment, the percentage of G0/ G1phase（43.03 ±1.45, t =9.49, P 〈0.05） was significantly increased. Conclusion MHBst155 protein can shorten the G0/ G1 phase of cell cycle, and improve the proliferation ability of cells. The mechanism may be associated with MHBst155 inducing abnormal methylation of cell growth regulatory genes.