本文選題：干擾素-α　切入點(diǎn)：肝非實(shí)質(zhì)細(xì)胞　出處：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)是一種嚴(yán)重威脅人類(lèi)健康的重要病原體。干擾素α (interferon α, IFN-α)是宿主抵抗病原入侵的天然免疫防線中一種重要細(xì)胞因子,也是目前臨床上治療慢性乙型肝炎(chronic hepatitis B, CHB)的一線藥物,在病人中有30-40%的應(yīng)答率。然而,目前IFN-α在體內(nèi)抗HBV的具體機(jī)制尚未完全闡明,同時(shí)國(guó)內(nèi)外研究證實(shí)HBV可通過(guò)多種機(jī)制抑制干擾素的產(chǎn)生、削弱效應(yīng)通路來(lái)拮抗干擾素效應(yīng)；體外或者HBV嵌合小鼠內(nèi)實(shí)驗(yàn)也證實(shí)IFN-α直接抑制肝實(shí)質(zhì)細(xì)胞(parenchymal cells)中HBV復(fù)制的能力并不強(qiáng),提示在體內(nèi)干擾素還可能通過(guò)其它途徑發(fā)揮抑制病毒復(fù)制的作用。 外體(exosomes)是一類(lèi)由細(xì)胞主動(dòng)分泌的大小介于30-100nm,內(nèi)含蛋白質(zhì)、RNA和脂質(zhì)的微囊結(jié)構(gòu),廣泛存在于各種體液中。已報(bào)道外體能介導(dǎo)細(xì)胞間的通信,外體內(nèi)含物包有大量功能性的：mRNA和microRNA等,通過(guò)水平轉(zhuǎn)移到鄰近或遠(yuǎn)處的靶細(xì)胞,參與調(diào)控多種重要的細(xì)胞生理活動(dòng)。本實(shí)驗(yàn)室李建華博士發(fā)現(xiàn)IFN-α能作用于肝非實(shí)質(zhì)細(xì)胞(non-paranchymal cells,包括肝竇內(nèi)皮細(xì)胞和巨噬細(xì)胞等),通過(guò)其分泌的外體在細(xì)胞間傳遞抗病毒分子,這些外體被HBV感染肝細(xì)胞內(nèi)化后可重塑肝細(xì)胞抗病毒狀態(tài),表明這可能是IFN-α發(fā)揮抗HBV作用的一個(gè)重要機(jī)制。那么IFN-α且體調(diào)節(jié)肝非實(shí)質(zhì)細(xì)胞所分泌外體中哪些mRNAs和]miRNAs差異分子,這些分子具有哪些功能,是否與抗病毒效果相關(guān),以及這些分子是否與臨床IFN-α療效有關(guān)聯(lián),亟待進(jìn)一步深入研究。 應(yīng)用全人類(lèi)基因組生物芯片檢測(cè)發(fā)現(xiàn)干擾素處理組與未處理組的肝竇內(nèi)皮細(xì)胞(liver sinusoidal endothelial cell, LSEC)所分泌外體所攜帶的miRNA和]mRNA全基因組表達(dá)譜差異(3組獨(dú)立重復(fù)),有1548種]mRNA和22種miRNAs有至少1.5倍以上顯著差異(Fold change, FC≥1.5, p0.05),其中共618種mRNA和3種miRNAs有1.5倍顯著上調(diào)。逆轉(zhuǎn)錄熒光定量PCR (qRT-PCR)經(jīng)隨機(jī)檢測(cè)11種上調(diào)mRNA和3種上調(diào)miRNA的表達(dá),結(jié)果與芯片檢測(cè)的基本一致,驗(yàn)證了LSEC芯片結(jié)果的可靠性。為了解供體細(xì)胞與其外體之間的表達(dá)差異,我們同時(shí)檢測(cè)了IFN-α處理前后LSEC供體細(xì)胞中上述14種分子的表達(dá),結(jié)果發(fā)現(xiàn)供體細(xì)胞與所分泌外體中分子的上調(diào)水平不一致,提示外體包含的分子具有特定的選入機(jī)制。我們又?jǐn)U展檢測(cè)了另一種重要的肝非實(shí)質(zhì)細(xì)胞,單核巨噬細(xì)胞來(lái)源的THP-1細(xì)胞本身及所分泌的外體中上述分子的表達(dá),其結(jié)果基本與LSEC中的趨勢(shì)一致,進(jìn)一步表明特定RNA分子存在于IFN-αα處理的肝非實(shí)質(zhì)細(xì)胞所分泌的外體中,可能是一種廣泛性的機(jī)制。 通過(guò)生物信息學(xué)方法分析在IFN-a處理后差異上調(diào)的618個(gè)mRNA中,發(fā)現(xiàn)有I型干擾素通路分子和49種干擾素調(diào)節(jié)相關(guān)基因(interferon regulated genes, IRGs)的富集,差異上調(diào)的3種miRNA通過(guò)計(jì)算機(jī)預(yù)測(cè)表明能參與多種細(xì)胞生物學(xué)功能,主要與細(xì)胞代謝通路相關(guān)。這些眾多的上調(diào)分子為進(jìn)一步篩選具有天然抗HBV活性分子提供一個(gè)較可靠數(shù)據(jù)庫(kù)。初步驗(yàn)證上述基因中的14種分子是否具有抗HBV活性,結(jié)果發(fā)現(xiàn)CALHM1, FABP5, DDIT3, miR-638, miR4-284轉(zhuǎn)染對(duì)上清HBsAg的表達(dá)量有顯著抑制(ELISA檢測(cè),p0.01); CALHM1, miR-4284對(duì)HBeAg也有顯著抑制(ELISA檢測(cè),p0.01)。 為了驗(yàn)證細(xì)胞實(shí)驗(yàn)結(jié)果與臨床干擾素治療慢性乙肝(CHB)病人樣本中的相關(guān)性,在34例長(zhǎng)效干擾素(PEG-IFN-a)治療有早期快速病毒學(xué)應(yīng)答(Early virological response, EVR)和40例PEG-IFN-a治療無(wú)EVR(對(duì)照組)的CHB病人血清樣本中,用Exo-quickTM法提取血漿外體,qRT-PCR比較上述14種mRNA和miRNA差異分子的水平變化。發(fā)現(xiàn)在有EVR應(yīng)答CHB病人中,第3個(gè)月較0個(gè)月血漿外體中的rniR-638、miR-4284、 miRNA-1260以及IFITM mRNA分子的相對(duì)升高倍數(shù),較無(wú)EVR病人有顯著升高(p0.01)。且IFITM的升高倍數(shù)與有EVR應(yīng)答病人的HBV DNA拷貝數(shù)的下降成相關(guān)性(相關(guān)系數(shù)為0.4045,p0.001),提示了外體中這些mRNA和miRNA分子作為指示IFN-αα應(yīng)答效果生物標(biāo)志物(biomarker)的可能性。 綜上所述,本研究分析了IFN-a誘導(dǎo)肝非實(shí)質(zhì)細(xì)胞(肝竇內(nèi)皮細(xì)胞和單核巨噬細(xì)胞)所分泌外體中l(wèi)nRNAs和niRNAs表達(dá)譜差異及部分上調(diào)分子的抗病毒活性,有助于了解IFN-a通過(guò)外體在機(jī)體內(nèi)微環(huán)境中發(fā)揮抗病毒作用的具體分子機(jī)制。通過(guò)在臨床PEG-IFN-a治療有無(wú)EVR應(yīng)答的慢性乙肝患者中進(jìn)一步驗(yàn)證部分差異分子表達(dá),為進(jìn)一步臨床研發(fā)基于血漿外體包含的mRNA和miRNA分子預(yù)測(cè)干擾素療效的生物標(biāo)志物提供了線索。
[Abstract]:Hepatitis B virus (hepatitis B, virus, HBV) is an important pathogen of serious threat to human health. Interferon alpha (interferon alpha, alpha IFN-) is an important cytokine in innate immunity of host defense against pathogen invasion, is currently the treatment of chronic hepatitis B (chronic hepatitis, B, CHB) of the line the drug, in patients with 30-40% response rate. However, the specific mechanism of IFN- alpha in vivo anti HBV has not been fully elucidated, and the domestic and foreign research confirmed that HBV can inhibit interferon through a variety of mechanisms have weakened the effect to antagonize the effect of interferon pathway; in vitro experiments or HBV chimeric mice also demonstrated that IFN- a direct inhibition of liver parenchymal cells (parenchymal cells) in HBV replication ability is not strong, suggesting that inhibition of viral replication may also play a role in other ways in vivo interferon.
Foreign body (exosomes) is a kind of active secretion by cell size is 30-100nm, containing protein, RNA and lipid microcapsule structure, widely exists in various body fluids have been reported. Physical mediated intercellular communication, and the contents in the package has a variety of functions: mRNA and microRNA, to the target cells near or distant by horizontal transfer, participate in the regulation of many important physiological activities of cells. The laboratory of Dr. Li Jianhua found that IFN- alpha can function in liver nonparenchymal cells (non-paranchymal and cells, including liver sinusoidal endothelial cells and macrophages), through the secretion of body transfer antiviral molecules between cells, the body infected by HBV liver cells after internalization can reshape the liver cell antiviral state, suggesting that this may be an important mechanism to play the role of anti HBV alpha IFN- alpha and IFN-. Then the body regulating hepatic non parenchymal cells which mRNAs is secreted by the outer body What are the functions of these molecules and]miRNAs differential molecules? Whether they are related to the antiviral effect, and whether these molecules are related to the efficacy of IFN- alpha, need further study.
The application of whole human genome microarray detection found that interferon treatment group and untreated group of liver sinusoidal endothelial cells (liver sinusoidal endothelial cell, LSEC) is secreted by the body carried by the miRNA and]mRNA genome expression profiles (3 independent replicates), there are at least 1.5 times more significant differences between the 1548 and 22 mRNA. MiRNAs (Fold change, FC = 1.5, P0.05), in which a total of 618 mRNA and 3 miRNAs with 1.5 times increased significantly. RT PCR (qRT-PCR) by random to detect the expression of 11 up-regulated mRNA and 3 by miRNA, the results are basically consistent with the microarray results, verify the reliability of LSEC chip in order to understand the donor. Differential expression between cells and their outer body, we also detected before and after IFN- treatment of LSEC donor cells expression of these 14 molecules, and found increased levels of donor cells and the secretion of molecules of the bodies Is not the same, suggesting that molecular body has specific selection mechanism. We also extend the detection of another important hepatic non parenchymal cells, the expressions of monocyte macrophage derived THP-1 cells and secreted by the body of the results with LSEC, consistent with the trend, further show that alpha alpha exists in IFN- treated liver non parenchymal cells outside the body is secreted by specific RNA molecules may be a mechanism of universality.
閫氳繃鐢熺墿淇℃伅瀛︽柟娉曞垎鏋愬湪IFN-a澶勭悊鍚庡樊寮備笂璋冪殑618涓猰RNA涓，

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