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  本文選題：第一高變區(qū)　切入點：丙型肝炎病毒　出處：《河北聯(lián)合大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的丙型肝炎病毒（hepatitis C virus, HCV）是導致肝硬化、肝細胞癌等終末期肝病的主要治病因子，目前尚無針對丙型肝炎的疫苗及特效的治療藥物，明確HCV入胞機制對治療丙型肝炎提供了新思路。第一高變區(qū)（HVR1）位于HCV包膜E2蛋白N端，是介導HCV包膜E2蛋白與細胞受體SR-BI結合的關鍵區(qū)域。通過建立HVR1蛋白與Raji細胞和L-02細胞結合模型，探究影響HVR1蛋白與細胞結合的關鍵分子，并進一步探索HVR1蛋白與細胞結合對下游信號通路的影響。 方法原核表達純化HVR1蛋白，HVR1蛋白與Raji細胞、L-02細胞共同孵育后，再與標記FITC的HVR1抗體共同孵育后進行流式檢測。利用小干擾RNA方法沉默SR-BI基因，并通過qPCR和western-blot檢測沉默效果，SR-BI沉默后流式檢測HVR1蛋白與細胞結合效率。western-blot檢測HVR1蛋白與細胞結合后ERK、 JNK信號通路，LDL存在HVR1蛋白與L-02細胞結合后JNK信號通路變化。 結果HVR1蛋白濃度在200μg/ml時與Raji細胞和L-02細胞孵育30min，結合率分別達到52.4%和58.9%。LDL存在情況下HVR1蛋白與Raji細胞結合率未有明顯變化，但是HVR1蛋白與L-02細胞結合率上升至85%。SR-BI基因沉默后HVR1蛋白與Raji細胞和L-02細胞結合率分別下降了63%和66%。western-blot檢測發(fā)現(xiàn)HVR1蛋白可以激活L-02細胞的ERK和JNK信號通路，但在Raji細胞中未發(fā)生ERK和JNK信號通路的活化，并且LDL存在情況下可以明顯抑制L-02細胞中JNK信號通路。 結論SR-BI受體在介導HVR1蛋白與Raji細胞和L-02細胞結合的過程中具有重要作用，LDL可以增強HVR1蛋白與L-02細胞的結合效率，HVR1與L-02細胞結合后可激活細胞中ERK和JNK信號通路，但LDL存在時JNK信號通路被抑制，因此推斷在HCV感染細胞的過程中，通過HVR1與細胞表面SR-BI受體結合，在低密度脂蛋白的介導下可能利用肝細胞脂代謝途徑大量吸附于細胞表面，并進一步活化下游ERK信號通路，抑制JNK信號通路，進而對肝細胞的增殖分化產生一定的影響。
[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.
【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.63

【參考文獻】

相關期刊論文 前1條

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本文編號：1592697