干扰素-α诱导肝非实质细胞分泌外体（exosomes）mRNA和miRNA表达谱的初步研究

发布时间：2018-03-23 17:13

  本文选题：干扰素-α　切入点：肝非实质细胞　出处：《复旦大学》2013年硕士论文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)是一种严重威胁人类健康的重要病原体。干扰素α (interferon α, IFN-α)是宿主抵抗病原入侵的天然免疫防线中一种重要细胞因子,也是目前临床上治疗慢性乙型肝炎(chronic hepatitis B, CHB)的一线药物,在病人中有30-40%的应答率。然而,目前IFN-α在体内抗HBV的具体机制尚未完全阐明,同时国内外研究证实HBV可通过多种机制抑制干扰素的产生、削弱效应通路来拮抗干扰素效应；体外或者HBV嵌合小鼠内实验也证实IFN-α直接抑制肝实质细胞(parenchymal cells)中HBV复制的能力并不强,提示在体内干扰素还可能通过其它途径发挥抑制病毒复制的作用。 外体(exosomes)是一类由细胞主动分泌的大小介于30-100nm,内含蛋白质、RNA和脂质的微囊结构,广泛存在于各种体液中。已报道外体能介导细胞间的通信,外体内含物包有大量功能性的：mRNA和microRNA等,通过水平转移到邻近或远处的靶细胞,参与调控多种重要的细胞生理活动。本实验室李建华博士发现IFN-α能作用于肝非实质细胞(non-paranchymal cells,包括肝窦内皮细胞和巨噬细胞等),通过其分泌的外体在细胞间传递抗病毒分子,这些外体被HBV感染肝细胞内化后可重塑肝细胞抗病毒状态,表明这可能是IFN-α发挥抗HBV作用的一个重要机制。那么IFN-α且体调节肝非实质细胞所分泌外体中哪些mRNAs和]miRNAs差异分子,这些分子具有哪些功能,是否与抗病毒效果相关,以及这些分子是否与临床IFN-α疗效有关联,亟待进一步深入研究。 应用全人类基因组生物芯片检测发现干扰素处理组与未处理组的肝窦内皮细胞(liver sinusoidal endothelial cell, LSEC)所分泌外体所携带的miRNA和]mRNA全基因组表达谱差异(3组独立重复),有1548种]mRNA和22种miRNAs有至少1.5倍以上显著差异(Fold change, FC≥1.5, p0.05),其中共618种mRNA和3种miRNAs有1.5倍显著上调。逆转录荧光定量PCR (qRT-PCR)经随机检测11种上调mRNA和3种上调miRNA的表达,结果与芯片检测的基本一致,验证了LSEC芯片结果的可靠性。为了解供体细胞与其外体之间的表达差异,我们同时检测了IFN-α处理前后LSEC供体细胞中上述14种分子的表达,结果发现供体细胞与所分泌外体中分子的上调水平不一致,提示外体包含的分子具有特定的选入机制。我们又扩展检测了另一种重要的肝非实质细胞,单核巨噬细胞来源的THP-1细胞本身及所分泌的外体中上述分子的表达,其结果基本与LSEC中的趋势一致,进一步表明特定RNA分子存在于IFN-αα处理的肝非实质细胞所分泌的外体中,可能是一种广泛性的机制。 通过生物信息学方法分析在IFN-a处理后差异上调的618个mRNA中,发现有I型干扰素通路分子和49种干扰素调节相关基因(interferon regulated genes, IRGs)的富集,差异上调的3种miRNA通过计算机预测表明能参与多种细胞生物学功能,主要与细胞代谢通路相关。这些众多的上调分子为进一步筛选具有天然抗HBV活性分子提供一个较可靠数据库。初步验证上述基因中的14种分子是否具有抗HBV活性,结果发现CALHM1, FABP5, DDIT3, miR-638, miR4-284转染对上清HBsAg的表达量有显著抑制(ELISA检测,p0.01); CALHM1, miR-4284对HBeAg也有显著抑制(ELISA检测,p0.01)。 为了验证细胞实验结果与临床干扰素治疗慢性乙肝(CHB)病人样本中的相关性,在34例长效干扰素(PEG-IFN-a)治疗有早期快速病毒学应答(Early virological response, EVR)和40例PEG-IFN-a治疗无EVR(对照组)的CHB病人血清样本中,用Exo-quickTM法提取血浆外体,qRT-PCR比较上述14种mRNA和miRNA差异分子的水平变化。发现在有EVR应答CHB病人中,第3个月较0个月血浆外体中的rniR-638、miR-4284、 miRNA-1260以及IFITM mRNA分子的相对升高倍数,较无EVR病人有显著升高(p0.01)。且IFITM的升高倍数与有EVR应答病人的HBV DNA拷贝数的下降成相关性(相关系数为0.4045,p0.001),提示了外体中这些mRNA和miRNA分子作为指示IFN-αα应答效果生物标志物(biomarker)的可能性。 综上所述,本研究分析了IFN-a诱导肝非实质细胞(肝窦内皮细胞和单核巨噬细胞)所分泌外体中lnRNAs和niRNAs表达谱差异及部分上调分子的抗病毒活性,有助于了解IFN-a通过外体在机体内微环境中发挥抗病毒作用的具体分子机制。通过在临床PEG-IFN-a治疗有无EVR应答的慢性乙肝患者中进一步验证部分差异分子表达,为进一步临床研发基于血浆外体包含的mRNA和miRNA分子预测干扰素疗效的生物标志物提供了线索。
[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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