乙肝病毒感染过程中白细胞介素-32的表达调控和抗病毒反应机制研究

发布时间：2018-08-11 13:08

【摘要】：乙型肝炎是世界性的流行性疾病,在全球每年至少导致了一百万人的死亡,也是影响我国人民健康最大的问题之一。乙肝病毒感染是导致慢性肝炎、肝硬化和肝癌的重要因素之一。本研究致力于慢性乙型肝炎过程中乙肝病毒感染与一种重要的炎症基因白细胞介素-32(白介素-32,IL-32)的相互关系的探讨,旨在阐明IL-32被病毒诱导表达的调控机制,并探明这一炎症因子在病毒感染过程中发挥的作用。 IL-32曾经被命名为自然杀伤细胞转录本4(NK4),最近有报道认为它是一种重要的炎症因子。它主要由免疫细胞表达,在一些病变的炎症组织中也有表达。为了确定该炎症因子在乙型肝炎中是否发挥作用,我们首先检测了它在177例慢性乙型肝炎病人和40例健康人血清中的含量。数据显示IL-32在病人血清中的含量显著性地高于正常人。于是我们开展了进一步的检测,分析确定慢性乙型肝炎病人血清中高水平的IL-32由何种细胞和组织表达产生的。肝脏细胞是乙肝病毒主要的宿主细胞,同时也有大量的报道表明人外周血单个核细胞(PBMC)也可以被乙肝病毒低水平的感染.因此我们检测了IL-32在这两种组织细胞中的表达水平。肝脏组织切片的免疫组织化学检测显示,慢性乙型肝炎的肝组织切片(n=16)中的IL-32水平明显高于正常人肝组织切片(n=16)。针对PBMC的荧光定量PCR结果显示,慢性乙型肝炎病人PBMC (n=17)中IL-32的mRNA水平显著性地高于正常人(n=10)。在肝癌细胞系和免疫细胞系中,我们转染乙肝病毒表达质粒pHBV-1.3同样可以诱导IL-32mRNA和蛋白的表达水平。这说明在乙肝病毒慢性感染者体内,肝细胞和PBMC中的IL-32都被病毒感染激活表达,并且存在分泌型的IL-32γ的表达,这使得血清中的IL-32水平显著升高。为了研究乙肝病毒诱导IL-32表达转录调控的分子生物学机制,我们首先利用P-mach软件对该基因的启动子进行分析,结果显示在750bp的核心启动子区域内,存在多个顺式作用元件,包括三个NF-κB和一个CREB结合位点。我们构建了核心启动子的报告基因质粒,并针对以上位点对IL-32启动子进行截短和突变。将这些报告质粒和pHBV-1.3或空载体共转染,通过报告基因萤光素酶活性检测分析显示,乙肝病毒重要通过激活IL-32启动子NF-κB位点,从而诱导转录。NF-KB通路特异性的抑制剂BAY-11实验也表明该通路被抑制时,乙肝病毒诱导的内源性的IL-32的表达大部分被抑制。染色体免疫共沉淀(CHIP)实验结果显示,乙肝病毒极大地促进了转录因子P65在NF-κB位点的结合。因此,在转录水平乙肝病毒主要通过激活NF-KB通路诱导IL-32表达。我们克隆了IL-32的3’非翻译区(UTR)并构建到含有报告基因的克隆载体上,与pHBV-1.3共转染后,我们发现乙肝病毒能上调该UTR的活性。这暗示在乙肝病毒对IL-32表达的翻译水平具有正向调控作用。最近报道,细小RNA (microRNA, miRNA)能通过结合基因mRNA的3'UTR来调节mRNA稳定性和翻译活性,是细胞内重要的翻译水平调控因子。我们利用miRNA结合预测软件进行分析,预测出20多种miRNA可能结合在该UTR上。根据我们已开展的乙肝病毒感染细胞系的miRNA表达芯片筛选结果,并参考以前报道的乙型肝炎中miRNA表达的临床数据,我们从这20多种(?)niRNA中找到了4个与乙肝病毒感染相关,它们分别是miR-29a, miR-29b, miR-29c, miR-223随后,我们从公司购买了这四种人工合成的成熟的miRNA以及它们对应的抑制子(互补的miRNA),然后将它们与IL-323'-UTR报告质粒进行共转染。荧光素酶检测分析结果显示,在四种miRNA中只有miR-29b能显著地抑制IL-323'UTR活性,并且在四种抑制子中唯有miR-29b能增强IL-323'UTR活性。为了进一步确认miR-29b对IL-32表达的调控作用,我们转染miR-29b和它的抑制子,然后检测IL-32的表达。结果显示,(?)niR-29b不仅能影响IL-323'UTR活性,而且参与了调控细胞内源性IL-32的表达。之后,为了确认miR-29b在IL-323'UTR上的结合位点,我们将构建好的报告质粒依照软件软件预测的结合位点进行了定点突变。结果显示突变后的UTR活性不受miR-29b和它的抑制子的影响,从而证实了软件预测的结合位点。最后我们在细胞系和病人临床样本中检测了miR-29b在乙肝病毒感染时的表达水平。结果显示,无论是在细胞系中还是在病人的PBMC中,乙肝病毒感染均可抑制这—miRNA水平。为了研究在体内乙肝病毒感染时(?)miR-29b对IL-32表达的调控关系,我们对17份慢性乙肝病人PBMC样本和8份乙肝病毒感染的肝脏组织样本中的miR-29b和IL-32水平做相关性分析,结果显示两种RNA水平在PBMC和肝组织样本中均存在显著的负相关性。因此,研究者认为乙肝病毒通过抑制miR-29b水平从而促进IL-32的翻译,这是对这一炎症基因表达调控机制的重要补充。 IL-32在乙型肝炎中发挥的作用,尤其是对病毒感染和复制的影响是该研究工作的另一重要部分。鉴于报道在IL-32六种间接异构体中γ亚型具有最好的活性,我们在乙肝病毒表达细胞系HepG2.2.15中采用IL-32γ开展抗病毒实验。但是结果显示无论是IL-32γ转染过表达质粒还是添加人源重组蛋白,乙肝病毒DNA和蛋白抗原表达水平都无显著性改变。由于乙型肝炎病人血清中IL-32高水平的存在,启发了研究者采用模拟体内真实环境的模型开展抗病毒实验。我们首先从正常人和肝炎病人血液中分离PBMC并进行体外培养,然后用重组IL-32γ蛋白进行刺激,利用收集的细胞培养上清液培养HepG2.2.15.结果显示乙肝病毒的复制和表达被这种上清液显著地抑制。此外,这种细胞培养上清液对艾滋病毒、肠道病毒-71和丙肝病毒的复制有显著性的抑制作用。研究者认为这种具有广谱抗病毒作用的细胞培养上清中存在着有效抗病毒作用的分子。基于Ⅰ型和Ⅲ型干扰素(IFN)是目前发现的最有效的抗病毒因子,我们开展了这两种IFN的抗体中和实验。结果显示,IFN-λ1抗体中和的上清液的抗病毒效果完全消失。这暗示重组IL-32γ蛋白在PBMC培养体系中导致了大量IFN-λ1的表达并分泌到上清液中从而使其具有抗病毒作用。针对IFNs的荧光定量PCR实验证实了IL-32γ能在PBMC中诱导IFN-λ1的表达,而IFN-α和IFN-β的表达量没有显著性的改变。深入的研究发现IL-32γ能通过激活IFN-λ1启动子远端的一串独特的NF-κB位点簇从而选择性地大量诱导这一抗病毒因子的表达。因此,我们认为血清中的IL-32γ通过在PBMC中特异性地诱导IFN-λ1的表达从而发挥抗病毒作用,这也阐明了IL-32抗病毒的途径和机制。我们用研究IL-32同样的方法研究了IFN-λ1的3'UTR,筛选出并证明了miR-548能结合IFN-λ1mRNA3'UTR,从而调控基因表达。并且miR-548/抑制子能通过调控IFN-λ1的表达影响细胞内天然免疫反应。同时我们发现,病毒感染时(?)niR-548表达水平是被抑制的。这是对病毒感染诱导IFN-γ1及其介导的抗病毒反应机制的重要补充。
[Abstract]:Hepatitis B is a worldwide epidemic disease, which causes at least one million deaths worldwide every year. It is also one of the biggest problems affecting the health of our people. Hepatitis B virus infection is one of the important factors leading to chronic hepatitis, cirrhosis and liver cancer. The purpose of this study is to elucidate the regulatory mechanism of IL-32 expression induced by viruses and to explore the role of IL-32 in viral infection.
IL-32, once named natural killer cell transcript 4 (NK4), has recently been reported to be an important inflammatory factor. It is mainly expressed in immune cells and in inflammatory tissues of some lesions. Serum levels of IL-32 in patients with hepatitis and 40 healthy controls were significantly higher than those in normal controls. Further tests were carried out to determine which cells and tissues were responsible for the production of high levels of IL-32 in the serum of patients with chronic hepatitis B. Hepatocytes were the predominant hepatitis B virus. There are also numerous reports that human peripheral blood mononuclear cells (PBMCs) can also be infected with low levels of hepatitis B virus. Therefore, we examined the expression of IL-32 in these two tissues and cells. Immunohistochemical examination of liver tissue sections showed that IL-32 in liver tissue sections of chronic hepatitis B (n=16) was water. The level of IL-32 mRNA in PBMC (n=17) of patients with chronic hepatitis B was significantly higher than that in normal controls (n=10). In hepatocellular carcinoma cell lines and immune cell lines, the expression plasmid pHBV-1.3 could also induce IL-32 mRNA and protein. This indicates that both hepatocytes and IL-32 in PBMC are activated by hepatitis B virus infection and secretory IL-32 gamma is expressed, which makes the level of IL-32 in serum increase significantly.
To study the molecular biological mechanism of transcriptional regulation of hepatitis B virus-induced IL-32 expression, we first analyzed the promoter of the gene using P-mach software. The results showed that there were several cis-acting elements in the 750 BP core promoter region, including three NF-kappa B and one CREB binding site. These reporter plasmids were co-transfected with pHBV-1.3 or empty vector, and the luciferase activity assay showed that hepatitis B virus could induce transcription by activating the NF-kappa B site of IL-32 promoter. The BAY-11 experiment also showed that the endogenous IL-32 expression induced by hepatitis B virus was mostly inhibited when the pathway was inhibited. Chromosome immunoprecipitation (CHIP) assay showed that hepatitis B virus greatly promoted the binding of transcription factor P65 at the NF-kappa B site. Therefore, hepatitis B virus induced IL mainly by activating the NF-KB pathway at the transcriptional level. -32 expression.
We cloned the 3'untranslated region (UTR) of IL-32 and constructed a vector containing reporter gene. After co-transfection with pHBV-1.3, we found that hepatitis B virus could up-regulate the activity of UTR. This suggests that hepatitis B virus has a positive regulatory effect on the translation level of IL-32 expression. The 3'UTR of gene mRNA regulates the stability and translation activity of mRNA, which is an important intracellular translation level regulator. We used the microRNA binding prediction software to analyze and predict that more than 20 kinds of microRNAs may bind to the UTR. Based on the microarray screening results of hepatitis B virus-infected cell lines, we also referred to the previous reports. Four of the more than 20 kinds of (?) niRNA were found to be associated with hepatitis B virus infection. They were microRNAs - 29a, microRNAs - 29b, microRNAs - 29c, and microRNAs - 223. We co-transfected with IL-323'-UTR reporter plasmid. Luciferase assay showed that only microRNAs-29b significantly inhibited IL-323'UTR activity in the four microRNAs, and only microRNAs-29b enhanced IL-323' UTR activity in the four inhibitors. In order to further confirm the regulatory effect of microRNAs-29b on IL-32 expression, we transfected microRNAs-29b and its expression. The results showed that (?) niR-29b not only affected the activity of IL-323'UTR, but also regulated the expression of endogenous IL-32 in cells. The results showed that the UTR activity of the mutant was not affected by the microRNAs-29b and its inhibitors, thus confirming the binding sites predicted by the software. Finally, we detected the expression of microRNAs-29b in hepatitis B virus infection in cell lines and patient clinical samples. In order to study the relationship between the expression of IL-32 and microRNAs-29b during hepatitis B virus infection in vivo, we analyzed the correlation between the levels of microRNAs-29b and IL-32 in 17 PBMC samples from patients with chronic hepatitis B and 8 liver samples from patients with hepatitis B virus infection. Therefore, researchers believe that hepatitis B virus promotes the translation of IL-32 by inhibiting the level of microRNAs-29b, which is an important complement to the regulation mechanism of this inflammatory gene expression.
The role of IL-32 in hepatitis B, especially in viral infection and replication, is another important part of this study. In view of the reported activity of the six indirect isoforms of IL-32, we used IL-32 gamma to carry out antiviral experiments in the hepatitis B virus expression cell line HepG2.2.15. There was no significant change in the expression of HBV DNA and protein antigens, whether IL-32gamma was transfected or human recombinant protein was added. The presence of high levels of IL-32 in the serum of patients with hepatitis B inspired researchers to carry out antiviral experiments using a model simulating the real environment in vivo. HepG2.2.15 was cultured with the collected cell culture supernatant. The results showed that the replication and expression of hepatitis B virus were significantly inhibited by the supernatant. In addition, the cell culture supernatant of HIV, enterovirus-71 and hepatitis C was stimulated by recombinant IL-32 gamma protein. Researchers believe that there are effective antiviral molecules in the supernatant of this broad-spectrum antiviral cell culture. Based on the type I and type III interferon (IFN) which is the most effective antiviral factor found so far, we have carried out antibody neutralization experiments for both IFN. The antiviral effect of the supernatant neutralized by antibody to lambda-1 disappeared completely, suggesting that the recombinant IL-32gamma protein resulted in the expression of a large number of IFN-lambda-1 in the PBMC culture system and secreted into the supernatant, thus making it have antiviral effect. The fluorescence quantitative PCR assay for IFNs confirmed that IL-32gamma could induce the expression of IFN-lambda-1 in PBMC, while IFN-alpha and IFN-lambda- In-depth studies have shown that IL-32gamma can selectively induce the expression of this antiviral factor by activating a unique cluster of NF-kappa B sites at the distal end of the IFN-lambda 1 promoter. Antiviral effect, which also elucidates the pathway and mechanism of IL-32 antiviral.
We studied the 3'UTR of IFN-lambda 1 in the same way as IL-32. We screened and demonstrated that microRNAs-548 could bind to IFN-lambda 1 mRNA 3'UTR to regulate gene expression. This is an important complement to the mechanism of viral infection inducing IFN- gamma 1 and its mediated antiviral response.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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