本文關(guān)鍵詞：高表達(dá)β2糖蛋白Ⅰ在乙型肝炎病毒入侵肝細(xì)胞初始環(huán)節(jié)中的作用研究　出處：《吉林大學(xué)》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： β2糖蛋白I 乙型肝炎病毒 肝細(xì)胞 鈉離子牛磺膽酸共轉(zhuǎn)運(yùn)多肽 膜聯(lián)蛋白II

【摘要】：乙型肝炎病毒(HBV)感染是全球主要的公共健康問(wèn)題，超過(guò)20億人感染HBV，有3.5-4億人為慢性感染，每年50-100萬(wàn)人死于乙肝相關(guān)性終末期肝病。我國(guó)屬于HBV高流行區(qū)，HBsAg檢出率為10%，有9,300萬(wàn)慢性HBV感染者，每年約35萬(wàn)人死于乙肝相關(guān)性肝硬化和肝細(xì)胞癌。目前針對(duì)HBV感染的防治，主要依賴于嬰兒期乙肝疫苗的預(yù)防接種和感染者抑制病毒復(fù)制。然而，阻止HBV入侵肝細(xì)胞和誘導(dǎo)肝細(xì)胞增殖則是更有效的治療方案。迄今為止，HBV入侵肝細(xì)胞的分子機(jī)制仍不能明確。 目前，普遍認(rèn)為HBV入侵肝細(xì)胞是由病毒包膜蛋白HBsAg調(diào)節(jié)的。HBsAg蛋白包括大、中、小三種形式，其中，表面抗原大蛋白(LHBs)由前S1、前S2和S區(qū)編碼蛋白組成，中蛋白(MHBs)由前S2和S區(qū)編碼蛋白組成，小蛋白(SHBs)由S區(qū)編碼蛋白組成。大蛋白前S1區(qū)被認(rèn)為是HBV入侵肝細(xì)胞的關(guān)鍵部位，可以與HBV受體結(jié)合。近年來(lái)，已發(fā)現(xiàn)很多HBV結(jié)合伴侶，可能是HBV入侵肝細(xì)胞途徑中的相關(guān)蛋白或者在肝細(xì)胞膜上的受體，主要有：β2糖蛋白I(beta2-GPI)、膜聯(lián)蛋白V、膜聯(lián)蛋白II (annexin II, ANX2)、去唾液酸糖蛋白受體(ASGPR)、纖維連接蛋白(Fibronectin)、糖胺聚糖(GAG)、鱗狀細(xì)胞癌抗原(SCCA)、羥肽酶D等等。最近研究認(rèn)為，鈉離子�；悄懰峁厕D(zhuǎn)運(yùn)多肽(NTCP)是人類乙型和丁型肝炎病毒的功能性受體。HBV的宿主特異性和嗜肝性與肝細(xì)胞表面特異性受體有關(guān)，深入探索HBV究竟通過(guò)哪種途徑如何入侵肝細(xì)胞至關(guān)重要。 β2糖蛋白I是血漿中含量豐富的糖蛋白之一，主要由肝細(xì)胞合成，與HBsAg具有高親和結(jié)合的特性。近年來(lái)，關(guān)于beta2-GPI的研究主要集中于作為自身抗原或共因子，參與抗磷脂綜合征等自身免疫性疾病血栓事件的發(fā)生。但有趣的是，beta2-GPI是個(gè)易變的分子，它的構(gòu)象和功能隨著與其結(jié)合的分子或者復(fù)合物的變化而改變。Beta2-GPI有五個(gè)功能區(qū)組成，類似于補(bǔ)體調(diào)控蛋白，第五功能區(qū)結(jié)構(gòu)特殊，帶有高度正電荷區(qū)。電子顯微鏡觀察顯示，健康人血漿中beta2-GPI以非致病的環(huán)狀形式存在，即第一功能區(qū)和第五功能區(qū)結(jié)合。當(dāng)陰離子結(jié)構(gòu)與第五功能區(qū)結(jié)合，beta2-GPI由閉合的環(huán)狀開(kāi)放呈“S”型，以利于與其他分子結(jié)合，表現(xiàn)多樣的生物學(xué)活性。已有研究發(fā)現(xiàn)，HBsAg與beta2-GPI第五功能區(qū)結(jié)合，beta2-GPI的糖基不能改變其與HBsAg的親和能力，在HBV病毒復(fù)制活躍期，HBsAg與beta2-GPI結(jié)合能力增強(qiáng)，還鑒定出beta2-GPI與肝癌細(xì)胞SMMC-7721細(xì)胞膜上annexin II特異性結(jié)合。我們推測(cè)：以beta2-GPI為中介，通過(guò)HBV-beta2-GPI-ANX2路徑入侵肝細(xì)胞，成為乙肝病毒親嗜肝細(xì)胞的可能途徑之一。 本研究首先以L02、SMMC-7721、HepG2、HepG2.2.15、293T、CHO細(xì)胞為研究對(duì)象，采用western blot方法檢測(cè)不同細(xì)胞中beta2-GPI的表達(dá)，其中：肝癌細(xì)胞SMMC-7721用于前期實(shí)驗(yàn)；HepG2.2.15由HepG2衍生而來(lái)，為HBVDNA轉(zhuǎn)染HepG2的穩(wěn)轉(zhuǎn)細(xì)胞系，能持續(xù)、穩(wěn)定表達(dá)HBV成熟病毒顆粒和HBsAg、HBeAg；L02為永化化胎兒正常肝臟細(xì)胞，作為正常對(duì)照；人胚腎細(xì)胞HEK-293T和中國(guó)倉(cāng)鼠卵巢癌細(xì)胞CHO-K1具有較高轉(zhuǎn)染效率，293T細(xì)胞還多用于病毒包裝的研究。結(jié)果表明，HepG2.2.15細(xì)胞中beta2-GPI表達(dá)明顯高于L02、SMMC-7721和HepG2細(xì)胞，而293T和CHO細(xì)胞中表達(dá)很少。通過(guò)qRT-PCR方法檢測(cè)同源細(xì)胞系HepG2和HepG2.2.15中beta2-GPI mRNA水平，發(fā)現(xiàn)HepG2.2.15細(xì)胞中beta2-GPI mRNA水平也明顯升高。接下來(lái)，選取L02、HepG2、HepG2.2.15和293T細(xì)胞為研究對(duì)象，將不同濃度梯度HBsAg或（和）beta2-GPI蛋白(0-800ng mL-1)與細(xì)胞共孵育，ELISA檢測(cè)發(fā)現(xiàn)beta2-GPI能增強(qiáng)HBsAg與不同細(xì)胞表面的結(jié)合，尤其是與L02和293T細(xì)胞，但無(wú)beta2-GPI濃度依賴性。已知beta2-GPI能與細(xì)胞膜上annexin II結(jié)合，我們認(rèn)為HBsAg與不同細(xì)胞表面結(jié)合能力的差異也可能與annexin II有關(guān)。進(jìn)一步通過(guò)westernblot法檢測(cè)不同細(xì)胞中annexin II的表達(dá)，發(fā)現(xiàn)HepG2.2.15細(xì)胞中annexin II含量明顯低于L02、SMMC-7721、HepG2和293T細(xì)胞，CHO細(xì)胞中有少量表達(dá)。 進(jìn)一步研究HBV和HBsAg與beta2-GPI的相互作用，將HBV和HBsAg表達(dá)載體分別與beta2-GPI質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，發(fā)現(xiàn)HBV和LHBsAg直接增強(qiáng)beta2-GPI表達(dá)，MHBsAg和SHBsAg對(duì)beta2-GPI表達(dá)無(wú)影響，還發(fā)現(xiàn)beta2-GPI能抑制HBsAg分泌。此外，將HBV表達(dá)載體轉(zhuǎn)染HepG2細(xì)胞中，HBV能降低annexin II表達(dá)。采用細(xì)胞免疫標(biāo)記技術(shù)，通過(guò)共聚焦顯微鏡觀察發(fā)現(xiàn)，HepG2.2.15細(xì)胞中beta2-GPI分別與HBsAg和annexin II共定位于細(xì)胞質(zhì)，，annexin II與HBsAg也共定位于細(xì)胞質(zhì)；HepG2細(xì)胞中，beta2-GPI和NTCP共定位于細(xì)胞膜，beta2-GPI和annexin II共定位于細(xì)胞質(zhì)中。 綜上研究，HBV使beta2-GPI表達(dá)上調(diào)，高表達(dá)beta2-GPI促進(jìn)HBsAg與細(xì)胞表面結(jié)合，有利于病毒與NTCP受體結(jié)合，beta2-GPI與annexin II結(jié)合可能促進(jìn)病毒與細(xì)胞表面結(jié)合和膜融合的發(fā)生。此結(jié)論為深入探索HBV入侵肝細(xì)胞的分子機(jī)制開(kāi)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.62

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相關(guān)期刊論文 前2條

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