丙型肝炎病毒核心蛋白对人源肝细胞生物学行为的影响

The effects of hepatitis C virus core protein on biological behaviors of human hepatocytes

目的探讨丙型肝炎病毒(HCV)核心蛋白对人源肝细胞生物学行为的影响及其相关机制。方法表达HCV核心蛋白的真核表达质粒pcDNA3.1core转染人源永生化肝细胞QSG7701,建立稳定表达HCV核心蛋白的细胞株——QSG7701/core,利用生长曲线、平板克隆实验和流式细胞术等方法检测HCV核心蛋白对细胞细胞生物学行为的影响;细胞免疫组织化学检测其对活化的caspase3蛋白表达的影响;利用Western印迹从磷酸化和非磷酸化水平检测核心蛋白对丝裂原蛋白激酶(MAPK)的影响;运用报道基因和凝胶电泳阻滞迁移实验(EMSA)等方法检测转录激活因子1(AP1)、核因子κB(NFκB)等转录因子活性的改变。结果HCV核心蛋白可明显抑制人源永生化肝细胞QSG7701的增殖和凋亡;同时下调MAPK通路激酶磷酸化水平和AP1、NFκB等转录因子的活性。结论HCV核心蛋白对MAPK通路和AP1、NFκB等转录因子的活性的负调控可能是其抑制增殖和凋亡的主要机制,并在病毒持续感染和病变慢性化以及肝癌(HCC)发生过程中起重要作用。

Objective To investigate the effects of hepatitis C virus (HCV) core protein on the biological behaviors of human hepatocytes and their underlying mechanism.Methods A cell line expressing stably HCV core protein—QSG7701/core was constructed by transfecting the plasmid pcDNA3.1-core (expressing HCV core protein) into the human immortalized hepatocytes of the line QSG7701. The biological behaviors of these transfected cells were observed through plating-efficiency test, growth curve and flow cytometry (FCM). The association between HCV core protein and the expression of activated caspase-3 protein was evaluated by immunocytochemistry. The phosphorylation of mitogen-activate protein kinases (MAPKs) was detected with Western blotting. The activation of nuclear transcriptors AP-1, important effector molecule of MAPKs, and nuclear factor-κ binding (NF-κB) were evaluated with luciferase assays and electrophoretic mobility shift assay (EMSA). Results HCV core protein was expressed in the QSG7701/core cells and not in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. There were no significant differences in the expression levels of total P44/42~MAPK , p38~MAPK and JNK among the QSG7701/core cells, QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. The expression levels of phophorylated P44/42~MAPK , p38~MAPK and JNK in the QSG7701/core cells were significantly weaker than those in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. Plating efficiency test showed that the clone formation rate of the QSG7701/core cells was 32.25%, significantly lower than those of QSG7701/pcDNA3.1 and untransfected QSG7701 cells (47.5% and 42.5% respectively, both P<0.01). The growth curve showed that the multiplication time of the QSG7701/core cells was 36 hours, significantly longer than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (27 and 28 hours respectively). FCM showed that the apoptotic rate of the QSG770/1core was 1.04%, lower than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (1.68% and 3.7% respectively), and that the percentage of theQSG770/1core cells at the G_0/G_1 stage increased and those in the S stage decreased. Immunocytochemistry showed that the expression intensity of caspase-3 in the QSG7701/ core cells was significantly weaker than those of the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. Conclusion HCV core protein suppresses cell proliferation and apoptosis by downregμlating the phosphorylation of MAPKs and activating the transcriptors AP-1 and NF-κB, thus promoting the persistency of HCV infection which leads to chronic hepatitis C and hepatocellular cancer.