ENPP2在抑制HBV病毒复制中的作用及机制的初步研究

发布时间：2018-03-03 12:55

本文选题：慢性乙型肝炎　切入点：ENPP2　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:慢性乙型肝炎是一个极其严重并影响全人类健康的公共卫生问题。据统计,全球60亿人口中大约有20亿人有HBV病毒感染的病史,其中约有3-4亿人有HBV DNA病毒慢性感染的病史,而我国正是HBV病毒感染的高流行区。慢性乙型肝炎不经过有效治疗或者治疗效果不佳时,会逐步发展为肝衰竭、肝硬化甚至是原发性肝癌,而据统计每年大约有100万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝细胞癌(HCC),占全球疾病死亡率的第7位。目前慢性乙型肝炎主要的治疗主要是干扰素和核苷(酸)类似物药物治疗,虽然这些药物能够在一部分患者使HBV DNA的复制得到有效控制,但仍有更大一部分患者疾病逐渐进展,药物并不能很好的改善。究其原因除了个体的差异性外,主要还是乙肝发病机制的复杂化和多变化,而我们对这种变化的不清楚使得很多研究仍致力于对乙肝发病机制更深入的研究。本研究也是意在研究慢乙肝的发病机制。方法:本研究以肝癌细胞HepG2和在其基础上建立的能够复制HBV DAN病毒的慢乙肝肝癌细胞HepG2.2.15为研究对象,应用蛋白质组学的方法(同位素相对标记与绝对定量技术,ITRAQ)筛选可能涉及到与乙肝病毒复制有关的差异表达的分泌蛋白。应用实时荧光定量RT-PCR和蛋白免疫印迹Western Blot技术对筛选出明显有差异的蛋白进行验证。为了研究可能涉及的乙肝发病机制,本研究组选取ENPP2蛋白进一步研究。本研究组采用酶联免疫吸附试验Elisa、蛋白免疫印迹Western Blot等实验验证ENPP2。为了研究ENPP2影响HBV DNA复制机制,本研究组分别给予ENPP2重组蛋白、ENPP2 siRNA、ENPP2质粒、ENPP2抗体、LPA受体抑制剂处理HepG2.2.15细胞,检测ENPP2、和LPA受体的,RT-PCR实验检测干扰素通路的变化,罗氏实验检测HBV DNA、HBsAg、HBeAg含量的变化。结果:结合iTRAQ技术、相关生物学分析网站以及统计学,我们筛选出133个有意义的蛋白,对比Hep G2细胞无血清培养上清液,有55种蛋白表达上调,其余的78种则表达下调。通过实时荧光定量RT-PCR和蛋白免疫印迹Western Blot验证一些差异显著并与肝脏疾病相关的蛋白,其结果与iTRAQ结果一致。Elisa和Western Blot进一步明确了ENPP2在慢性乙型肝炎患者中血清中普遍升高。大量的细胞实验发现HBV DNA的复制可以诱导ENPP2的表达,且被诱导表达后的ENPP2呈现了对HBV复制的抑制作用,同时干扰素通路发生了协同性变化,LPA受体发挥了关键性作用。结论:ENPP2在慢性乙型肝炎患者中普遍高表达;且ENPP2可通过与LPA1-3受体作用抑制Hep G2.2.15细胞的HBV复制。
[Abstract]:Objective: chronic hepatitis B (CHB) is an extremely serious public health problem affecting the health of all mankind. According to statistics, about 2 billion out of 6 billion people in the world have a history of HBV infection. About 300 million of them have a history of chronic HBV DNA infection, and China is a highly endemic area for HBV infection. Chronic hepatitis B can develop into liver failure without effective treatment or poor treatment. Liver cirrhosis and even primary liver cancer. According to statistics, about 1 million people die every year from liver failure caused by HBV infection. Liver cirrhosis and primary hepatocellular carcinoma (HCC) account for the 7th highest mortality rate in the world. At present, the main treatment for chronic hepatitis B is interferon and nucleoside analogues. Although these drugs can effectively control the replication of HBV DNA in some patients, there are still a larger number of patients with progressive disease, and the drugs do not improve very well. Mainly because the pathogenesis of hepatitis B is complicated and varied. However, our lack of clarity about this change has led many studies to study the pathogenesis of hepatitis B. this study is also intended to study the pathogenesis of chronic hepatitis B. methods: this study is based on HepG2 and its base on liver cancer cells. On the basis of this study, HepG2.2.15 cell line, which can replicate HBV DAN virus, was used as the research object. Proteomics (isotopic relative labeling and absolute quantitative techniques) was used to screen secretory proteins that might be involved in differentially expressed HBV replication. Real-time fluorescent quantitative RT-PCR and Western blot Western were used to screen secretory proteins that might be related to HBV replication. In order to study the possible pathogenesis of hepatitis B, Blot technique was used to verify the screening of proteins with obvious differences. In order to study the effect of ENPP2 on the replication of HBV DNA, we selected ENPP2 protein for further study. In this study, HepG2.2.15 cells were treated with ENPP2 recombinant protein ENPP2 siRNA-ENPP2 plasmids and ENPP2 antibody against LPA receptor inhibitor. ENPP2and LPA receptor were detected by reverse transcription-polymerase chain reaction (RT-PCR) to detect the changes of interferon pathway, and the changes of HBeAg content in HBV DNA-HBs were detected by Roche assay. Results: combined with iTRAQ technique, HBeAg was detected in HepG2.2.15 cells. Related biological analysis sites and statistics, we screened 133 meaningful proteins, compared with Hep G2 cell serum-free culture supernatant, 55 proteins were up-regulated. The other 78 down-regulated proteins were identified by real-time fluorescent quantitative RT-PCR and Western blot Western Blot. The results were consistent with the results of iTRAQ. Elisa and Western Blot further confirmed that ENPP2 generally increased in serum of patients with chronic hepatitis B. A large number of cell experiments showed that the replication of HBV DNA could induce the expression of ENPP2. The induced expression of ENPP2 could inhibit the replication of HBV, and the synergistic change of interferon pathway might play a key role in the expression of ENPP2. Conclusion the expression of ENPP2 is generally high in patients with chronic hepatitis B. ENPP2 inhibited HBV replication in Hep G2.2.15 cells by interaction with LPA1-3 receptor.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.62

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