乙型肝炎病毒包膜蛋白诱导肝癌细胞系内质网应激的研究

Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus

目的探讨乙型肝炎病毒前-S缺失和野生型外膜大蛋白(LHBs)诱导内质网应激反应。方法运用脂质体转染技术将pc DNA3.1-S、pc DNA3.1-L、pc DNA3.1-L△30和pc DNA3.1-L△182等真核表达质粒转染至Huh7细胞,并应用q RT-PCR和Western blot方法检测转染细胞前-S1、前-S2、HBs Ag和GRP78的m RNA和蛋白表达水平。结果转染HBV包膜蛋白各组的HBs Ag蛋白相对表达量(分别为0.92、0.83、0.91、1.75、2.53和2.06)高于空白对照(0.00)和pc DNA3.1(+)转染组(0.00)(F=247.38、P=0.000);转染HBV包膜蛋白各组的GRP78的蛋白相对表达量(分别为1.4、1.47、1.55、1.75、1.8和1.9)高于空白对照(1.18)和pc DNA3.1(+)转染组(1.23)(F=11.623、P=0.000);且GRP78蛋白相对表达量与前-S1、前-S2抗原和HBs Ag表达量呈正相关(相关系数r分别为0.884、0.728和0.816)。结论前-S缺失型/野生型LHBs及HBs Ag均能诱导Huh7细胞发生内质网应激反应,提示其可能参与肝癌发生。

Objective To explore endoplasmic reticulum stress in Huh7 cells induced by wild type and pre-S mutant envelope proteins of HBV. Methods The recombinant eukaryotic expression vectors of pc DNA3.1-S, pc DNA3.1-L, pc DNA3.1-L△30 and pc DNA3.1-L△182 were transfected into Huh7 cells through liposome transfection, then the protein exrpression of pre-S1, pre-S2, HBs Ag and Grp78 were detected by Western blot, and Grp78 m RNA transcripts were detected by q RT-PCR. Results The relative expression of HBs Ag protein in Huh7 cells transfected with recombinant eukaryotic expression vectors（0.92, 0.83, 0.91, 1.75, 2.53 and 2.06, respectively） were significantly higher than that in blank control cells（0.00） and that in cells transfected with pc DNA3.1（＋）（0.00）（F = 247.38, P = 0.000）. The relative expression of GRP78 protein in Huh7 cells transfected with recombinant eukaryotic expression vectors（1.4, 1.47, 1.55, 1.75, 1.8 and 1.9, respectively） were significantly higher than that in blank control cells（1.18） and that in cells transfected with pc DNA3.1（＋）（1.23）（F = 11.623, P = 0.000）. The relative expression of GRP78 protein had opositive correlation with that of pre-S1, pre-S2 and HBs Ag（r = 0.884, 0.728 and 0.816, respectively）. Conclusions Expression of envelope proteins of HBV could induce endoplasmic reticulum stress, indicating they might involve in hepatocarcinogenesis.