丙型肝炎病毒非结构蛋白4B对肝癌细胞增殖的影响

The effect of hepatitis C virus NS4B to the proliferation of HepG2 cells

目的探讨丙型肝炎病毒非结构蛋白4B(HCV-NS4B)对肝癌细胞增殖及增殖细胞核抗原(PCNA)和周期素D1(cyclinD1)蛋白表达的影响。方法采用构建好的HCV-NS4B表达载体,以脂质体转染法转染HepG2细胞。RT-PCR、琼脂糖凝胶电泳证实HCV-NS4B在HepG2细胞稳定表达。MTT法绘制生长曲线,观察NS4B对肝细胞生长的影响;流式细胞仪检测细胞周期的情况;免疫细胞化学法检测PCNA和cyclinD1的表达。结果转染后,生长曲线显示NS4B可促进肝细胞生长;细胞周期检测显示转染NS4B的HepG2细胞进入S期和G2/M期增多(P<0.05),处于G0/G1期细胞降低(P<0.05)。PCNA和cyclinD1的表达均较空白载体组升高(P<0.05)。结论HCV-NS4B可干扰肝癌细胞周期,促进肝癌细胞增殖,其作用与上调PCNA、cyclinD1表达有关。

Objective To investigate the effect of hepatitis C virus NS4B （HCV-NS4B） to the proliferation and the expression of PCNA and cyclinD1 in HepG2 cells. Methods The recombinant vector PCXN2-NS4B expressing HCV NS4B gene was transfected into HepG2 cells using liposomal transfection reagent kit. The expression of the transfected NS4B gene was determined by RT-PCR. The growth curve was made by MTF. The cell cycle was detected by flow cytome try. Semiquantitative immunocytochemistry assays was used to analyze the expression of PCNA and cyclinD1. Results The results from RT-PCR analysis demonstrated that there was the replication of HCV-NS4B gene in the transfected HepG2 cells. The growth curve showed that HCV-NS4B could improve the proliferation of HepG2 cells. The ratios of HepG2 cells in S and G2/M stages were significantly higher in NS4B transfected group than those in control group （ P 〈 0.05 ）. The ex pression of PCNA and cyclinD1 in NS4B transfected group were significantly higher than those in control group （P 〈 0.05）. Conclusion HCV-NS4B can promote the proliferation of hepatoma carcinoma cells through increasing the expres sion of PCNA and cyclinD1.