【摘要】： 丙型病毒性肝炎是嚴(yán)重危害人類健康的傳染病。全球丙肝病毒(HCV)的感染人數(shù)約為一億七千萬，占世界人口總數(shù)的1—3％。在感染的人群中，約80％轉(zhuǎn)為慢性，20％發(fā)展為肝硬化和肝細(xì)胞癌；我國的HCV感染者占人口總數(shù)的3.2％，估計(jì)感染者總數(shù)約為4000萬。 自從1989年HCV cDNA首次被克隆成功至今，對HCV的分子生物學(xué)研究取得了突飛猛進(jìn)的發(fā)展。目前已明確，HCV為單股正鏈RNA病毒，屬黃病毒科，全長約9600個(gè)核苷酸，ORF區(qū)編碼3010個(gè)氨基酸的多聚蛋白前體，經(jīng)過宿主和病毒本身基因編碼的蛋白酶裂解為十個(gè)功能性片段，其中四個(gè)為結(jié)構(gòu)蛋白，分別是核心蛋白C、包膜蛋白E1、E2以及P7；六個(gè)為非結(jié)構(gòu)蛋白，分別是NS2、NS3、NS4A、NS4B、NS5A和NS5B。盡管HCV的基本結(jié)構(gòu)已經(jīng)清楚，，但是HCV的防治仍然是世界性的難題，α干擾素和病毒唑聯(lián)合治療是唯一的治療方法，但這種治療方法只對不到50％的患者顯效，且具有費(fèi)用高、易復(fù)發(fā)和副作用多等缺陷；尤其是我國流行的HCV-Ⅱ／1b型，對α干擾素的治療應(yīng)答最低，因此發(fā)展治療和預(yù)防性丙肝疫苗，對于預(yù)防HCV感染和控制感染者的病情發(fā)展，都具有重要的意義。 HCV NS3蛋白具有絲氨酸蛋白酶和解旋酶活性，參與病毒蛋白翻譯后加工，為病毒復(fù)制所必須，針對NS3蛋白的T細(xì)胞應(yīng)答與自限性感染關(guān)系密切。目前公認(rèn)位于NS3區(qū)的1248—1261位氨基酸殘基為CD4~+Th1細(xì)胞表位，而且該表位對于MHC限制性具有廣譜特異性。在此之前的研究已經(jīng)以BALB／c小鼠為實(shí)驗(yàn)動(dòng)物模型，以MHCⅡ抗原提呈通路中的分子伴侶恒定鏈(invariant chain，Ii)為載體，用HCV-NS3的1248-1261表位替換Ii鏈的CLIP編碼片段構(gòu)建了內(nèi)源性靶向基因疫苗，并在細(xì)胞水平研究了所構(gòu)建的基因疫苗與抗原提呈分子之間的相互作用，用所構(gòu)建的疫苗對荷瘤小鼠進(jìn)行了免疫治療并取得了好的效果。 本研究構(gòu)建了內(nèi)源性靶向基因疫苗的體外轉(zhuǎn)錄載體，在體外轉(zhuǎn)錄成mRNA，同時(shí)用小鼠的骨髓細(xì)胞體外誘導(dǎo)培養(yǎng)，成功的培養(yǎng)出小鼠骨髓來源的樹突細(xì)胞，通過電轉(zhuǎn)的方法將內(nèi)源性靶向基因疫苗mRNA導(dǎo)入小鼠樹突細(xì)胞(dendritic cell，DC)內(nèi)，研究內(nèi)源性靶向基因疫苗對DC表面分子的表達(dá)及細(xì)胞因子的分泌的影響。并用混合淋巴細(xì)胞實(shí)驗(yàn)來研究內(nèi)源性靶向基因疫苗是否影響DC對T細(xì)胞誘導(dǎo)分化功能。研究結(jié)果如下： 1．內(nèi)源性基因疫苗可有效地刺激樹突細(xì)胞成熟，增加表面協(xié)同刺激分子CD80、CD86、I-A~d的表達(dá)。 2．內(nèi)源性基因疫苗可有效地誘導(dǎo)DC分泌細(xì)胞因子IL-12、IFN-γ。 3．經(jīng)內(nèi)源性基因疫苗負(fù)載的DC可有效的刺激CD4~+T細(xì)胞增殖，并誘導(dǎo)其向Th1方向分化。 綜上所述，用以Ii鏈作為載體針對HCV-NS3的Th1表位內(nèi)源性靶向基因疫苗修飾DC后，可增強(qiáng)DC的抗原提呈功能，能有效地激活CD4~+T細(xì)胞。這種方法有望為HCV的防治提供一種可行的途徑，為下一步構(gòu)建靶向樹突細(xì)胞的內(nèi)源性基因疫苗奠定基礎(chǔ)，在治療性HCV基因疫苗的開發(fā)研究中具有良好的應(yīng)用前景。
[Abstract]:Type C viral hepatitis is an infectious disease that is seriously harmful to human health. The worldwide prevalence of hepatitis C virus (HCV) is about 100 million, accounting for 1-3 per cent of the world's total population. In the infected population, about 80% to chronic and 20% developed into liver cirrhosis and hepatocellular carcinoma; HCV-infected people in China account for 3.2% of the total population, and the total number of people infected is estimated to be about 40 million. The molecular biology of HCV has been studied since the first cloning of the HCV cDNA in 1989. The present invention has shown that HCV is a single-stranded positive-chain RNA virus, which is a single-stranded positive-chain RNA virus, which is a polyprotein precursor of the total length of 9600 nucleotides and the ORF region encoding 3010 amino acids, and the protease which is encoded by the host and the virus itself is cracked into ten functional fragments, Four of which are structural proteins, respectively, are core protein C, envelope protein E1, E2 and P7, and six are non-structural proteins, and are NS2, NS3, NS4A and NS4B, respectively. NS5A and NS5B. Although the basic structure of HCV is clear, the prevention and treatment of HCV is still a worldwide problem. The combination therapy of interferon and virotherapy is the only treatment method, but the treatment method is effective only for less than 50% of patients, and has high cost and easy recovery. There are many defects such as hair and side effects, especially the prevalence of HCV-II/ 1b in China, and the treatment response of interferon is the lowest. Therefore, the development of treatment and preventive hepatitis C vaccine can be used to prevent HCV infection and to control the development of the infected people. The HCV NS3 protein has the activity of serine protease and helicase, participates in the post-translational processing of the viral protein, The T cell response of the 3-protein is closely related to the self-infection. It is recognized that the 1248-1261-position amino acid residue in the NS3 region is the CD4 ~ + Th1 cell epitope. And the epitope has broad-spectrum specificity for MHC restriction. The prior research has been used as an experimental animal model in BALB/ c mice, and the molecular chaperone constant chain (Ii) in the path of the MHC II antigen is taken as a carrier, and the C of the Ii chain is replaced by the 1248-1261 epitope of the HCV-NS3. The LIP coding fragment constructs the endogenous targeting gene vaccine and studies the interaction between the constructed gene vaccine and the antigen-presenting molecule at the cell level, using the constructed vaccine pair In this study, the in vitro transcription vector of the endogenous target gene vaccine was constructed and the mRNA was transcribed in vitro, and the mice were used in the same time. The bone marrow cells were cultured in vitro, and the dendritic cells from the bone marrow of the mouse were successfully cultured. The endogenous target gene vaccine mRNA was introduced into the mouse dendritic cell (DC) by the method of electrospinning. The effect of the target gene vaccine on the expression of DC surface and the secretion of cytokines. to study endogenous Whether the target gene vaccine affects the DC-to-T cell-induced differentiation function. The results are as follows:1. The endogenous gene vaccine can effectively stimulating the maturation of the dendritic cells, increasing the surface synergistic stimulation molecules CD80, CD86, I-A- 2. The endogenous gene vaccine can effectively induce the secretion of the cytokines IL-12, I, The DC of the endogenous gene vaccine load can stimulate the proliferation of CD4 ~ + T cells and induce it to differentiate into the Th1 direction. The method provided by the invention has the functions of enhancing the antigen extraction function of the DC and effectively activating the CD4 + T cell by using the Ii chain as a vector to modify the DC of the endogenous target gene of the Th1 epitope of the HCV-NS3,
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 徐全壹;“寒淫”致病及轉(zhuǎn)歸的轉(zhuǎn)錄組與代謝組實(shí)驗(yàn)研究[D];成都中醫(yī)藥大學(xué);2012年

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