乙型肝炎病毒蛋白对肝细胞炎性反应及调控机制研究

发布时间：2018-01-16 03:28

本文关键词：乙型肝炎病毒蛋白对肝细胞炎性反应及调控机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：天然免疫反应系统在抵抗乙型肝炎病毒(HBV)感染中发挥重要作用。作为肝脏最主要的组成,肝细胞在肝脏免疫调控中可能发挥了主导作用。本课题研究了 HBV蛋白表达对肝细胞/肝癌细胞炎性反应的调控及其作用机制,为阐明HBV蛋白与肝细胞在肝脏疾病发生发展过程中的作用提供了研究基础。研究方法1、构建HBV蛋白表达重组载体,转染肝细胞/肝癌细胞。通过荧光PCR研究肝细胞/肝癌细胞内炎性效应分子在转录水平上的表达变化趋势,利用酶联免疫吸附试验(ELISA)和免疫印迹试验(Western Blot)来研究炎性效应分子在蛋白水平的表达变化情况;2、利用Western Blot和免疫荧光试验,借助特异的信号通路抑制剂,研究HBV蛋白引起的炎性效应中涉及到的信号通路及其作用;3、构建HBV核心抗原(HBc)稳转细胞株,检测HBc在细胞增殖中的作用;4、转染siRNA研究肝癌细胞内HMGB1在HBc促细胞增殖过程中的作用,结合Western Blot技术,研究涉及到的信号通路。研究结果1、HBV 大蛋白(LHBs)和 HBc 能够上调 AIM2、IL-1β、IL-18、IL-6和TN αmRNA等炎性基因的表达;中蛋白(MHBs)能够上调AIM2、IL-6和TNFα mRNA的表达;小蛋白(SHBs)除引起TNFα表达量上调外,对其它基因的表达无影响。在SMMC-7721、L-02和Huh7细胞中,LHBs、HBc和MHBs组IL-6表达量上调且在24小时达到高值。在HepG2和SMMC-7721细胞中,MHBs和HBc组TNFα表达量在转染6小时时达到高值,随后下降,在Huh7和L-02细胞中,变化趋势与此相反。在Huh7细胞中,MHBs上调HMGB1的表达,且在24小时达到高值,在L-02和SMMC-7721细胞中,随时间延长,MHBs组HMGB1的表达量逐渐下降;2、MHBs激活MAPKp38/NF-KB以促进IL-6的表达和分泌,这种效应依赖于内质网压力;在Huh7细胞中,MHBs活化NF-κB来上调HMGB1的表达,且引起HMGB1从细胞核转移到胞质;3、HBc通过激活MAPK p38、ERK1/2和NF-κB来促进IL-6的表达和分泌,但此效应与内质网压力无关;在Huh7细胞中,HBc激活NF-κB以促进HMGB1的表达,但不会引起HMGB1从细胞核到胞质的移位;HBc促进Huh7细胞的增殖,HMGB1 siRNA抑制HBc的促增殖作用,同时也抑制HBc对ERK1/2的激活,但对MAPKp38信号通路无影响。研究结论1、同一 HBV蛋白在不同细胞株中引起的效应不一致,同一细胞株对不同HBV蛋白的反应也不同。总体来说,LHBs、MHBs和HBc会引起炎性基因表达的变化;除TNFα外,SHBs对肝细胞/肝癌细胞内其它炎性基因的表达无显著影响;2、MHBs诱导产生内质网压力,激活p38/NF-κB通路,上调IL-6的表达与分泌,增强HMGB1的表达,并导致其从核转移到胞质,表明,MHBs主要作为肝细胞/肝癌细胞的压力性刺激因子起作用;3、HBc 激活 MAPK p38、ERK1/2 和 NF-κB,上调 IL-6 和 HMGB1 的表达,促进肝癌细胞增殖,表明,HBc作为促炎因子和促增殖因子,具有多重作用。
[Abstract]:Innate immune response system plays an important role in resisting hepatitis B virus (HBV) infection and is the most important component of liver. Hepatocytes may play a leading role in the regulation of liver immunity. In this study, we studied the regulation of HBV protein expression on the inflammatory response of hepatocytes / hepatoma cells and its mechanism. In order to elucidate the role of HBV protein and hepatocytes in the development of liver disease. 1. Construct recombinant vector of HBV protein expression. Transfection of hepatocytes / hepatoma cells. The expression trend of inflammatory effector molecules in hepatocytes / hepatoma cells at the transcriptional level was studied by fluorescence PCR. Enzyme linked immunosorbent assay (Elisa) and Western blot were used to study the expression of inflammatory effector molecules at protein level. 2. By using Western Blot and immunofluorescence assay, the signal pathway involved in the inflammatory effect of HBV protein and its role were studied with the help of specific signal pathway inhibitors. 3. HBV core antigen (HBC) was constructed and transformed into cells stably, and the role of HBc in cell proliferation was detected. 4. The role of HMGB1 in the proliferation of hepatoma cells induced by HBc was studied by transfection of siRNA, combined with Western Blot technique. Results 1. HBc and HBc can up-regulate IL-18. Expression of inflammatory genes such as IL-6 and TN 伪 mRNA; The expression of IL-6 and TNF 伪 mRNA was up-regulated by MHBsAg. The expression of TNF 伪 in SMMC-7721 L-02 and Huh7 cells was not affected, except for the up-regulation of TNF 伪 expression in SMMC-7721 L-02 and Huh7 cells. The expression of IL-6 was up-regulated in HBc and MHBs groups and reached a high level at 24 hours, in HepG2 and SMMC-7721 cells. The expression of TNF 伪 in MHBs and HBc groups reached a high level at 6 hours after transfection, and then decreased. In Huh7 and L-02 cells, the change trend was reversed. MHBs upregulated the expression of HMGB1 and reached a high level at 24 hours. In L-02 and SMMC-7721 cells, the expression of HMGB1 in L-02 and SMMC-7721 cells decreased with time. 2MHBs activate MAPKp38/NF-KB to promote the expression and secretion of IL-6, which depends on endoplasmic reticulum pressure; In Huh7 cells, MHBs activate NF- 魏 B to up-regulate the expression of HMGB1 and induce the transfer of HMGB1 from the nucleus to the cytoplasm. 3HBC stimulated the expression and secretion of IL-6 by activating MAPK p38 ERK1 / 2 and NF- 魏 B, but this effect was not related to endoplasmic reticulum pressure. In Huh7 cells, HBC activates NF- 魏 B to promote the expression of HMGB1, but does not induce the translocation of HMGB1 from nucleus to cytoplasm. HBc promoted the proliferation of Huh7 cells. HMGB1 siRNA inhibited the proliferation of HBc and the activation of ERK1/2 by HBc. Conclusion 1. The effects of the same HBV protein on different cell lines were not consistent. The response of the same cell line to different HBV proteins was also different. Generally speaking, MHBs and HBc of LHBs could induce the change of inflammatory gene expression. The expression of other inflammatory genes in hepatocytes / hepatoma cells was not significantly affected by SHBs except TNF 伪. 2MHBs induced endoplasmic reticulum pressure, activated p38 / NF- 魏 B pathway, up-regulated the expression and secretion of IL-6, enhanced the expression of HMGB1, and led to its transfer from nucleus to cytoplasm. MHBs mainly acts as a stress-stimulating factor in hepatocytes / hepatoma cells. 3HBC activated MAPK p38 ERK1 / 2 and NF- 魏 B, upregulated the expression of IL-6 and HMGB1, and promoted the proliferation of hepatoma cells. HBc, as a proinflammatory and proliferative factor, has multiple effects.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.62

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