高表达β2糖蛋白Ⅰ在乙型肝炎病毒入侵肝细胞初始环节中的作用研究

发布时间：2018-01-18 01:00

本文关键词：高表达β2糖蛋白Ⅰ在乙型肝炎病毒入侵肝细胞初始环节中的作用研究　出处：《吉林大学》2014年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(HBV)感染是全球主要的公共健康问题，超过20亿人感染HBV，有3.5-4亿人为慢性感染，每年50-100万人死于乙肝相关性终末期肝病。我国属于HBV高流行区，HBsAg检出率为10%，有9,300万慢性HBV感染者，每年约35万人死于乙肝相关性肝硬化和肝细胞癌。目前针对HBV感染的防治，主要依赖于婴儿期乙肝疫苗的预防接种和感染者抑制病毒复制。然而，阻止HBV入侵肝细胞和诱导肝细胞增殖则是更有效的治疗方案。迄今为止，HBV入侵肝细胞的分子机制仍不能明确。目前，普遍认为HBV入侵肝细胞是由病毒包膜蛋白HBsAg调节的。HBsAg蛋白包括大、中、小三种形式，其中，表面抗原大蛋白(LHBs)由前S1、前S2和S区编码蛋白组成，中蛋白(MHBs)由前S2和S区编码蛋白组成，小蛋白(SHBs)由S区编码蛋白组成。大蛋白前S1区被认为是HBV入侵肝细胞的关键部位，可以与HBV受体结合。近年来，已发现很多HBV结合伴侣，可能是HBV入侵肝细胞途径中的相关蛋白或者在肝细胞膜上的受体，主要有：β2糖蛋白I(beta2-GPI)、膜联蛋白V、膜联蛋白II (annexin II, ANX2)、去唾液酸糖蛋白受体(ASGPR)、纤维连接蛋白(Fibronectin)、糖胺聚糖(GAG)、鳞状细胞癌抗原(SCCA)、羟肽酶D等等。最近研究认为，钠离子牛磺胆酸共转运多肽(NTCP)是人类乙型和丁型肝炎病毒的功能性受体。HBV的宿主特异性和嗜肝性与肝细胞表面特异性受体有关，深入探索HBV究竟通过哪种途径如何入侵肝细胞至关重要。 β2糖蛋白I是血浆中含量丰富的糖蛋白之一，主要由肝细胞合成，与HBsAg具有高亲和结合的特性。近年来，关于beta2-GPI的研究主要集中于作为自身抗原或共因子，参与抗磷脂综合征等自身免疫性疾病血栓事件的发生。但有趣的是，beta2-GPI是个易变的分子，它的构象和功能随着与其结合的分子或者复合物的变化而改变。Beta2-GPI有五个功能区组成，类似于补体调控蛋白，第五功能区结构特殊，带有高度正电荷区。电子显微镜观察显示，健康人血浆中beta2-GPI以非致病的环状形式存在，即第一功能区和第五功能区结合。当阴离子结构与第五功能区结合，beta2-GPI由闭合的环状开放呈“S”型，以利于与其他分子结合，表现多样的生物学活性。已有研究发现，HBsAg与beta2-GPI第五功能区结合，beta2-GPI的糖基不能改变其与HBsAg的亲和能力，在HBV病毒复制活跃期，HBsAg与beta2-GPI结合能力增强，还鉴定出beta2-GPI与肝癌细胞SMMC-7721细胞膜上annexin II特异性结合。我们推测：以beta2-GPI为中介，通过HBV-beta2-GPI-ANX2路径入侵肝细胞，成为乙肝病毒亲嗜肝细胞的可能途径之一。本研究首先以L02、SMMC-7721、HepG2、HepG2.2.15、293T、CHO细胞为研究对象，采用western blot方法检测不同细胞中beta2-GPI的表达，其中：肝癌细胞SMMC-7721用于前期实验；HepG2.2.15由HepG2衍生而来，为HBVDNA转染HepG2的稳转细胞系，能持续、稳定表达HBV成熟病毒颗粒和HBsAg、HBeAg；L02为永化化胎儿正常肝脏细胞，作为正常对照；人胚肾细胞HEK-293T和中国仓鼠卵巢癌细胞CHO-K1具有较高转染效率，293T细胞还多用于病毒包装的研究。结果表明，HepG2.2.15细胞中beta2-GPI表达明显高于L02、SMMC-7721和HepG2细胞，而293T和CHO细胞中表达很少。通过qRT-PCR方法检测同源细胞系HepG2和HepG2.2.15中beta2-GPI mRNA水平，发现HepG2.2.15细胞中beta2-GPI mRNA水平也明显升高。接下来，选取L02、HepG2、HepG2.2.15和293T细胞为研究对象，将不同浓度梯度HBsAg或（和）beta2-GPI蛋白(0-800ng mL-1)与细胞共孵育，ELISA检测发现beta2-GPI能增强HBsAg与不同细胞表面的结合，尤其是与L02和293T细胞，但无beta2-GPI浓度依赖性。已知beta2-GPI能与细胞膜上annexin II结合，我们认为HBsAg与不同细胞表面结合能力的差异也可能与annexin II有关。进一步通过westernblot法检测不同细胞中annexin II的表达，发现HepG2.2.15细胞中annexin II含量明显低于L02、SMMC-7721、HepG2和293T细胞，CHO细胞中有少量表达。进一步研究HBV和HBsAg与beta2-GPI的相互作用，将HBV和HBsAg表达载体分别与beta2-GPI质粒共转染293T细胞，发现HBV和LHBsAg直接增强beta2-GPI表达，MHBsAg和SHBsAg对beta2-GPI表达无影响，还发现beta2-GPI能抑制HBsAg分泌。此外，将HBV表达载体转染HepG2细胞中，HBV能降低annexin II表达。采用细胞免疫标记技术，通过共聚焦显微镜观察发现，HepG2.2.15细胞中beta2-GPI分别与HBsAg和annexin II共定位于细胞质，，annexin II与HBsAg也共定位于细胞质；HepG2细胞中，beta2-GPI和NTCP共定位于细胞膜，beta2-GPI和annexin II共定位于细胞质中。综上研究，HBV使beta2-GPI表达上调，高表达beta2-GPI促进HBsAg与细胞表面结合，有利于病毒与NTCP受体结合，beta2-GPI与annexin II结合可能促进病毒与细胞表面结合和膜融合的发生。此结论为深入探索HBV入侵肝细胞的分子机制开辟了新思路和新方向。
[Abstract]:Hepatitis B virus (HBV) infection is a major public health problem worldwide, more than 2 billion people are infected with HBV, 3.5-4 million people with chronic infection, 50-100 million people die each year from HBV related end-stage liver disease. Our country belongs to the area of high HBV prevalence, the detection rate of HBsAg was 10%, 93 million persons infected with chronic HBV, about 350 thousand people each year died of HBV related cirrhosis and hepatocellular carcinoma. For the prevention of HBV infection, vaccination and inhibit the replication of the virus infection mainly depends on infant hepatitis B vaccine. However, stop the HBV invasion of liver cells and liver cell proliferation induced by treatment is more effective. So far, the molecular mechanism of HBV invasion of liver cells still not clear.
At present, HBV is generally considered invasion of liver cells by virus envelope protein HBsAg regulates.HBsAg protein, including large, mistress forms, the large surface protein (LHBs) by S1, S2 and S before the formation of encoding protein, protein (MHBs) by S2 and S before encoding protein. Small protein (SHBs) by S encoding proteins. Protein S1 is considered to be the key parts of the HBV invasion of liver cells, can be combined with the HBV receptor. In recent years, have been found in many HBV binding partners, may be related to protein HBV pathway in liver cell invasion or receptor in liver cell membrane the main are: Beta 2 glycoprotein I (beta2-GPI), annexin V, annexin II (annexin II ANX2), asialoglycoprotein receptor (ASGPR), fibronectin (Fibronectin), glycosaminoglycan (GAG), squamous cell carcinoma antigen (SCCA), carboxypeptidase D and so on. Recent studies suggest that Niu Huangdan, sodium ion Total acid (NTCP) is a human transporter polypeptide of hepatitis B and hepatitis D virus functional receptor.HBV host specificity and hepatotropism and liver specific surface receptors, explore whether HBV channels through which how the invasion of liver cells is essential.
Beta 2 glycoprotein I is one of the most abundant glycoproteins in plasma, mainly synthesized by liver cells, has the characteristics of high affinity binding with HBsAg. In recent years, the researches on beta2-GPI mainly focus on as a self antigen or co factor involved in the antiphospholipid syndrome and autoimmune diseases such as thrombotic events. But interesting is that beta2-GPI is a molecular variable, its molecular conformation and function with its binding or complex changes of.Beta2-GPI is composed of five functional areas, similar to the complement regulatory protein structure, fifth functional areas, with a high positive charge region. Electron microscopy showed that in healthy human plasma by non beta2-GPI the pathogenic cyclic form, namely the first functional area and fifth functional areas combined. When combined with anion structure and fifth functional areas, beta2-GPI showed "S" type is a closed circular opening, to help with the Other molecules, has various biological activities. It has been found that the combination of HBsAg and beta2-GPI fifth functional areas, affinity glycosylation of beta2-GPI cannot change with HBsAg, active replication in HBV virus, HBsAg and beta2-GPI binding ability enhancement, also identified with annexin II specific beta2-GPI and SMMC-7721 cells membrane. We speculated that mediated by beta2-GPI through the HBV-beta2-GPI-ANX2 path invasion of liver cells, become one of the possible ways of hepatitis B virus tropism of liver cells.
In this study, L02, SMMC-7721, HepG2, HepG2.2.15293T, CHO cells as the research object, using the Western blot method to detect the expression of beta2-GPI in different cells in the SMMC-7721 cells: for pre experiment; HepG2.2.15 is derived from HepG2, HBVDNA and HepG2 transfected cell lines stably transfected, sustained, stable expression of HBV mature virus particles and HBsAg, HBeAg; L02 of Yong Hua normal fetal liver cells, as normal control; human embryonic kidney cell HEK-293T and China hamster ovarian carcinoma CHO-K1 cells with high transfection efficiency, 293T cells for virus packaging research. The results showed that the expression of beta2-GPI was significantly higher than that of L02 in HepG2.2.15 cells, SMMC-7721 cells and HepG2 cells, and 293T and CHO cells was detected by the qRT-PCR method. A few homologous beta2-GPI level of mRNA cell lines HepG2 and HepG2.2.15, beta2-G in HepG2.2.15 cells The PI level of mRNA was significantly increased. Next, select L02, HepG2, HepG2.2.15 and 293T cells as the research object, the different concentration of HBsAg or (and) beta2-GPI protein (0-800ng mL-1) and cells were incubated, ELISA assay showed that beta2-GPI could enhance the binding between HBsAg and cell surface, especially with L02 and 293T cells no, but beta2-GPI concentration dependent. With the known beta2-GPI cell membrane annexin binding to II, we think that HBsAg with different cell surface binding ability differences may also be related to annexin II. Further by Westernblot method to detect the expression of annexin in different cell lines II, annexin found II content in HepG2.2.15 cells was significantly lower than that of L02, SMMC-7721, HepG2 and 293T cells expressed in CHO cells.
The interaction of HBV and HBsAg and further study of beta2-GPI, HBV and HBsAg respectively with beta2-GPI expression vector plasmid were transfected into 293T cells, HBV and LHBsAg directly enhance the expression of beta2-GPI, MHBsAg and SHBsAg had no effect on the expression of beta2-GPI, also found that beta2-GPI can inhibit the secretion of HBsAg. In addition, the HBV expression vector was transfected into HepG2 cells, HBV annexin can reduce the expression of II. The cell immune markers by confocal microscopy revealed that HepG2.2.15 cells in beta2-GPI and HBsAg respectively and annexin II were localized in the cytoplasm, annexin II and HBsAg were localized in the cytoplasm; HepG2 cells, beta2-GPI and NTCP were localized in the cell membrane, beta2-GPI and annexin II were located in the in the cytoplasm.
In conclusion, HBV up-regulated the expression of beta2-GPI, high expression of beta2-GPI promotes the combination of HBsAg and the cell surface for virus combined with NTCP receptor, beta2-GPI and annexin combined with II may accelerate the occurrence of virus and cell surface binding and membrane fusion. This conclusion provides a new idea and new directions for further exploring the molecular mechanism of HBV invasion of liver cells.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62

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