丙型肝炎病毒NS4B调控肝细胞增殖的相关机制研究

Regulating the proliferation of hepatocyte by hepatitis C virus non-structural protein 4B:study of related mechanisms

目的研究丙型肝炎病毒非结构蛋白4B(HCV-NS4B)对肝细胞IGF-Ⅱ、P16表达的影响,探讨HCV-NS4B对肝细胞增殖的影响及相关机制。方法转染载体PCXN2或PCXN2-NS4B至LO2肝细胞中,以正常LO2肝细胞为空白对照利用MTT法绘制空白载体PCXN2组及PCXN2-NS4B组细胞生长曲线,流式细胞仪检测各组细胞周期;利用放射免疫法测定各组的IGF-Ⅱ水平,RT-PCR检测P16的表达情况。结果 (1)PCXN2-NS4B质粒转染入LO2细胞并有稳定表达。(2)PCXN2-NS4B组与PCXN2组比较,细胞生长速度增加。(3)PCXN2组G0/G1期细胞比例高于PCXN2-NS4B组,而S期及G2/M细胞比例低于后者(P<0.05或P<0.01)。(4)空白对照组、PCXN2两组IGF-Ⅱ水平差异无统计学意义(P>0.05),但均显著低于PCXN2-NS4B组(P<0.01)。(5)空白对照组、PCXN2两组P16表达差异无统计学意义,但均显著高于PCXN2-NS4B组(P<0.01)。结论 PCXN2-NS4B质粒转染入肝细胞后有稳定表达,HCV-NS4B可能通过促进IGF-Ⅱ的表达及抑制P16表达而促进肝细胞异常增殖。

Objective To study the effects of hepatitis C virus non-structural protein 4B（HCV-NS4B）on the expression of insulin-like growth factor Ⅱ（IGF-Ⅱ）and P16,and to investigate its effects on hepatocellular proliferation and possible mechanisms.Methods LO2 hepatocytes were randomly divided into three groups according to different treatment.In PCXN2 group and PCXN2-NS4B group,they were transfected with empty vector PCXN2 and recombinant plasmid PCXN2-HCV-NS4B,respectively.Those without transfection were served as blank control group.MTT method was used to measure the OD value of cultured hepatocytes in PCXN2 group and PCXN2-NS4B group,and the findings were delineated as cell growth curve.Cell cycle,IGF-Ⅱ level and P16 expression was detected by flow cytometry,radioimmunoassay and reverse transcription PCR（RT-PCR）,respectively.Results（1）Recombinant plasmid PCXN2-NS4B was successfully transfected into LO2 hepatocytes and was steadily expressed.（2） Compared with PCXN2 group,cell growth curve demonstrated that cell growth increased in PCXN2-NS4B group（P0.01）.（3） The rate of G0/ G1 was higher,but that of G2/M or S phase was lower in PCXN2 group than that in PCXN2-NS4B group（P0.05 or P0.01）.（4）IGF-Ⅱlevels in blank control group and PCXN2 group increased significantly compared with PCXN2-NS4B group（P0.01）although no significant difference was found between them.（5） P16 expression between blank control group and PCXN2 group was not significantly different,but both groups showed higher P16 expression than PCXN2-NS4B group（P0.01）.Conclusion Recombinant plasmid PCXN2-NS4B is steadily expressed in LO2 hepatocytes after transfection.HCV-NS4B may promote abnormal proliferation of hepatocyte by enhancing IGF-Ⅱ expression and inhibiting P16 expression.