丙型肝炎病毒高变区1介导HCV感染细胞机制的初步研究

发布时间：2018-03-10 09:16

本文选题：第一高变区　切入点：丙型肝炎病毒　出处：《河北联合大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的丙型肝炎病毒（hepatitis C virus, HCV）是导致肝硬化、肝细胞癌等终末期肝病的主要治病因子，目前尚无针对丙型肝炎的疫苗及特效的治疗药物，明确HCV入胞机制对治疗丙型肝炎提供了新思路。第一高变区（HVR1）位于HCV包膜E2蛋白N端，是介导HCV包膜E2蛋白与细胞受体SR-BI结合的关键区域。通过建立HVR1蛋白与Raji细胞和L-02细胞结合模型，探究影响HVR1蛋白与细胞结合的关键分子，并进一步探索HVR1蛋白与细胞结合对下游信号通路的影响。方法原核表达纯化HVR1蛋白，HVR1蛋白与Raji细胞、L-02细胞共同孵育后，再与标记FITC的HVR1抗体共同孵育后进行流式检测。利用小干扰RNA方法沉默SR-BI基因，并通过qPCR和western-blot检测沉默效果，SR-BI沉默后流式检测HVR1蛋白与细胞结合效率。western-blot检测HVR1蛋白与细胞结合后ERK、 JNK信号通路，LDL存在HVR1蛋白与L-02细胞结合后JNK信号通路变化。结果HVR1蛋白浓度在200μg/ml时与Raji细胞和L-02细胞孵育30min，结合率分别达到52.4%和58.9%。LDL存在情况下HVR1蛋白与Raji细胞结合率未有明显变化，但是HVR1蛋白与L-02细胞结合率上升至85%。SR-BI基因沉默后HVR1蛋白与Raji细胞和L-02细胞结合率分别下降了63%和66%。western-blot检测发现HVR1蛋白可以激活L-02细胞的ERK和JNK信号通路，但在Raji细胞中未发生ERK和JNK信号通路的活化，并且LDL存在情况下可以明显抑制L-02细胞中JNK信号通路。结论SR-BI受体在介导HVR1蛋白与Raji细胞和L-02细胞结合的过程中具有重要作用，LDL可以增强HVR1蛋白与L-02细胞的结合效率，HVR1与L-02细胞结合后可激活细胞中ERK和JNK信号通路，但LDL存在时JNK信号通路被抑制，因此推断在HCV感染细胞的过程中，通过HVR1与细胞表面SR-BI受体结合，在低密度脂蛋白的介导下可能利用肝细胞脂代谢途径大量吸附于细胞表面，并进一步活化下游ERK信号通路，抑制JNK信号通路，进而对肝细胞的增殖分化产生一定的影响。
[Abstract]:Objective Hepatitis C virus (HCV) is the main therapeutic factor of liver cirrhosis, hepatocellular carcinoma and other end-stage liver diseases. It is clear that the mechanism of HCV entry provides a new idea for the treatment of hepatitis C. the first hypervariable region (HVR1) is located at the N-terminal of E2 protein of HCV capsule. HCV E2 protein is the key region that mediates the binding of HCV envelope E2 protein to cell receptor SR-BI. By establishing a model of HVR1 binding to Raji cells and L-02 cells, the key molecules affecting the binding of HVR1 protein to cells were explored. The effect of HVR1 protein binding with cells on downstream signaling pathway was further explored. Methods the purified HVR1 protein was incubated with Raji cell line L-02, then incubated with the HVR1 antibody labeled with FITC. The SR-BI gene was silenced by small interfering RNA method. QPCR and western-blot were used to detect the silencing effect. After SR-BI silencing, the binding efficiency of HVR1 protein to cells was detected by flow cytometry. Western blot was used to detect the changes of JNK signal pathway after HVR1 protein binding to cells. JNK signal pathway was changed after HVR1 protein combined with L-02 cells. Results HVR1 protein was incubated with Raji cells and L-02 cells at the concentration of 200 渭 g / ml for 30 min. The binding rates of HVR1 protein and Raji cells were 52.4% and 58.9% respectively. There was no significant change in the binding rate between HVR1 protein and Raji cells. However, the binding rate of HVR1 protein to L-02 cells increased to 85k.SR-BI gene silencing, and the binding rate of HVR1 protein to Raji cells and L-02 cells decreased by 63% and 66.western-blot, respectively. It was found that HVR1 protein could activate ERK and JNK signaling pathway in L-02 cells. However, ERK and JNK signaling pathways were not activated in Raji cells, and the JNK signaling pathway in L-02 cells was significantly inhibited in the presence of LDL. Conclusion SR-BI receptor plays an important role in mediating the binding of HVR1 protein to Raji cells and L-02 cells. LDL can enhance the binding efficiency of HVR1 protein to L-02 cells. HVR1 and L-02 cells can activate the ERK and JNK signaling pathway after binding to L-02 cells. However, in the presence of LDL, the JNK signaling pathway was inhibited. It was inferred that during the process of HCV infection, the HVR1 could bind to the SR-BI receptor on the cell surface, which might be adsorbed on the cell surface by the lipid metabolism pathway of hepatocytes mediated by low density lipoprotein. Further activation of downstream ERK signaling pathway, inhibition of JNK signaling pathway, and then affect the proliferation and differentiation of hepatocytes.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.63

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