【摘要】：丙型肝炎病毒(HCV)是一種帶有包膜的黃病毒科家族RNA病毒,它包含一條長為9600個堿基的正義單鏈RNA基因組。目前全世界有超過1億7000萬的人遭受著HCV慢性感染,并且它也是導(dǎo)致慢性肝病甚至肝癌的一個主要原因。由于丙肝所引發(fā)的疾病負(fù)擔(dān)和人員死亡正成倍增長,丙肝和艾滋病一樣,都嚴(yán)重摧殘著人類的身心健康,導(dǎo)致嚴(yán)重的公共衛(wèi)生安全和社會問題。因此,進(jìn)一步展開丙肝病毒感染引起肝癌的內(nèi)在分子機(jī)制的研究顯得尤為迫切和重要。 本研究中,我們發(fā)現(xiàn)在HCV感染的病人的外周血單個核細(xì)胞中,信號轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化子-3(STAT3),基質(zhì)金屬蛋白酶-2(MMP-2)以及B細(xì)胞淋巴因子-2(Bcl-2)的mRNA表達(dá)水平相對于健康人群都有顯著的上調(diào)。通過體外JFH-1亞型的HCV活病毒感染Huh7.5.1細(xì)胞實(shí)驗(yàn),結(jié)果表明：HCV能刺激STAT3,MMP-2, Bcl-2以及細(xì)胞外信號調(diào)節(jié)激酶(ERK),C-Jun氨基末端激酶(JNK)的表達(dá),并呈現(xiàn)出時間和劑量依賴效應(yīng).我們進(jìn)一步對HCV基因組的10個蛋白進(jìn)行功能性篩選,發(fā)現(xiàn)非結(jié)構(gòu)蛋白4B(NS4B)能激活STAT3信號通路,上調(diào)MMP-2以及Bcl-2的表達(dá),同時也能抑制細(xì)胞信號轉(zhuǎn)導(dǎo)抑制因子-3(SOCS3)的表達(dá)。通過細(xì)胞核抽提,熒光共聚焦以及過表達(dá)實(shí)驗(yàn),我們發(fā)現(xiàn)NS4B蛋白能促進(jìn)STAT3的入核,顯著上調(diào)STAT3的磷酸化水平。當(dāng)我們分別用激酶(PKC, JNK, ERK)的干擾RNA轉(zhuǎn)染細(xì)胞后,發(fā)現(xiàn)STAT3的磷酸化水平受到抑制,MMP-2以及Bcl-2的表達(dá)水平也相應(yīng)下調(diào)。證明PKC, JNK以及ERK在NS4B激活STAT3信號通路中扮演重要角色。此外,由于已有研究表明NS4B的C末端結(jié)構(gòu)域(CTD)與HCV的復(fù)制相關(guān),能和細(xì)胞因子相互作用,我們進(jìn)一步研究發(fā)現(xiàn)NS4B的CTD能顯著激活STAT3, ERK以及JNK,上調(diào)MMP-2, BCL-2的表達(dá),其中從第227到第250這24個氨基酸區(qū)域尤為重要,同時我們通過點(diǎn)突變實(shí)驗(yàn)證實(shí)第237,239,245這三個氨基酸殘基的改變,能極大的抑制NS4B上調(diào)STAT3通路,表明第237,239,245這三個氨基酸殘基對NS4B的功能的發(fā)揮起著重要作用。 由于MMP-2和BCL-2與細(xì)胞轉(zhuǎn)化,腫瘤形成有重要關(guān)系,本研究闡明了一種新的HCV感染的致病機(jī)制,為臨床疾病診斷以及新藥靶標(biāo)設(shè)計提供了新的思路和實(shí)驗(yàn)依據(jù)。
[Abstract]:Hepatitis C virus (HCV) is a family of RNA virus with envelope. It contains a sense single-stranded RNA genome with a length of 9600 bases. At present, more than 100 million people worldwide suffer from chronic HCV infection, and it is also a major cause of chronic liver disease and even liver cancer. As the burden of disease and death caused by hepatitis C is increasing exponentially, both hepatitis C and AIDS seriously destroy the physical and mental health of human beings, leading to serious public health safety and social problems. Therefore, it is urgent and important to further study the intrinsic molecular mechanism of liver cancer caused by hepatitis C virus infection. In this study, we found that the expression of signal transduction and transcriptional activator 3 (STAT3), matrix metalloproteinase-2 (MMP-2) and B-cell lymphoid factor-2 (Bcl-2) in peripheral blood mononuclear cells (PBMC) of patients with HCV infection were significantly up-regulated than those of healthy controls. In vitro, HCV subtype of JFH-1 subtype of HCV infected Huh7.5.1 cells. The results showed that HCV could stimulate the expression of STAT3,MMP-2, Bcl-2 and extracellular signal-regulated kinase (ERK) C-Jun amino-terminal kinase (JNK) in a time-and dose-dependent manner. We further screened 10 proteins in the HCV genome and found that non-structural protein 4B (NS4B) could activate the STAT3 signaling pathway, up-regulate the expression of MMP-2 and Bcl-2, and inhibit the expression of cell signal transduction inhibitor 3 (SOCS3). Through nuclear extraction, fluorescence confocal and overexpression experiments, we found that NS4B protein can promote the entry of STAT3 and significantly up-regulate the phosphorylation level of STAT3. After transfection of RNA with kinase (PKC, JNK, ERK) interference, we found that the phosphorylation level of STAT3 was inhibited and the expression of MMP-2 and Bcl-2 were down-regulated. It is demonstrated that PKC, JNK and ERK play an important role in the activation of STAT3 signaling pathway by NS4B. In addition, it has been shown that the C-terminal domain (CTD) of NS4B is related to the replication of HCV and can interact with cytokines. We further found that CTD of NS4B can significantly activate STAT3, ERK and JNK, upregulate the expression of MMP-2, BCL-2. The 24-amino acid region from 227th to 250th is particularly important, and we confirmed by point mutation experiment that the change of the three amino acid residues of 237239245 can significantly inhibit the up-regulation of STAT3 pathway by NS4B. These three amino acid residues 237239245 play an important role in the function of NS4B. Because MMP-2 and BCL-2 have important relationship with cell transformation and tumor formation, this study clarifies a new mechanism of HCV infection, and provides new ideas and experimental basis for clinical disease diagnosis and new drug target design.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R373

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前1條

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