【作者】 刘霞；

【导师】 朱强；

【作者基本信息】 山东大学 ， 内科学（消化系病）（专业学位）， 2019， 博士

【摘要】 乙型肝炎病毒(Hepatitis B Virus,HBV)感染引起的肝硬化是威胁全人类健康的主要疾病之一,全球HBV感染者约20亿人,我国约1 5～25%患者因HBV感染相关性疾病而死亡。目前认为乙型肝炎肝硬化(Hepatitis B Virus related Cirrhosis,HBV-related Cirrhosis)发生发展中存在多阶段免疫功能紊乱,早期及时采取干预措施,肝纤维化过程可以被逆转。人类HBV感染不同阶段获得性及固有性免疫功能变化的分子机制各不相同,慢性乙型病毒性肝炎(Chronic Hepatitis,CHB)以病毒特异性的适应性免疫功能失活为主,而乙肝肝硬化则会出现固有免疫功能的紊乱,探索乙肝肝硬化固有免疫紊乱调控机制及调控靶点对研究阻断疾病进程的新技术及临床新药研发有着重大意义。T淋巴细胞免疫球蛋白黏蛋白3(T-cell immunoglobulin and mucin domain-containing protein 3,Tim-3)最早发现表达在辅助I型T淋巴细胞(Help T cell 1,Th1)表面,被认为是Th1特异性表面蛋白分子,后来研究证明Tim-3还可表达在多种免疫细胞如自然杀伤细胞(natural killer cell,NK cell)、树突状细胞(Dentritic cell,DC)、CD8+T细胞和单核/巨噬细胞(Monocyte/Macrophage)等表面。Tim-3主要是通过与其配体半乳糖凝集素-9(galectin-9)结合发挥作用,可介导CHB患者体内CD8+T细胞功能抑制,促进其调亡。还有研究发现CHB患者外周血和肝组织中CD14+CD16+单核细胞亚群明显升高,通过分泌大量的促炎因子,刺激肝脏炎症持续存在,在CHB患者病情进展及转归中发挥重要作用。但是,Tim-3蛋白分子在乙肝肝硬化患者体内单核细胞亚群中的表达及功能目前尚不清楚,其是否参与HBV感染介导的肝硬化疾病进程,有待进一步研究。Tim-3是由Tim基因家族转录后形成的一类跨膜蛋白,存在着转录及转录后修饰水平、翻译及翻译后修饰水平的调控。非编码RNA(Non-coding RNA,ncRNA)是近年来新发现的一类小分子RNA,主要通过与靶基因序列结合参与调节靶基因转录及翻译水平的调控,主要包括microRNA(miRNA)和Long noncoding RNA(1ncRNA)等,microRNA与1ncRNA存在交互作用,通过调控靶基因蛋白表达,进一步调节基因蛋白功能。本课题从检测乙肝肝硬化患者外周血中不同亚群单核细胞Tim-3蛋白表达水平着手,证实Tim-3蛋白存在不同程度的表达差异,同时伴随着体内外使用LPS刺激单核细胞的多种炎症因子水平的差异。进一步地,我们从非编码RNA的角度挖掘到调控Tim-3基因的microRNA-545和1ncRNA Ftx,从而发现并证实了非编码RNA Ftx/microRNA-545参与对Tim-3的表达调控并影响多种炎症因子分泌这一重要调节路径,这一路径可能在乙肝肝硬化炎症进程中发挥重要作用。本课题分以下四部分展开相应研究:第一部分Tim-3蛋白在乙肝肝硬化患者外周血单核细胞中的表达研究研究目的:通过比较乙肝肝硬化患者与健康人群对照组外周血不同单核细胞亚群Tim-3蛋白的差异表达水平,从而初步判断Tim-3蛋白在乙肝肝硬化发生发展过程中发挥相应作用。研究方法:采集32名乙肝肝硬化患者(实验组)和36名健康志愿者(对照组)的新鲜外周抗凝血:①采用三色荧光全血染色法,用荧光素标记的抗CD14、抗CD16和抗Tim-3进行免疫荧光染色,应用流式细胞仪检测;②分离单核细胞,制作细胞石蜡蜡块,切片后进行免疫组织化学染色,检测Tim-3蛋白表达。结果:流式细胞仪检测和免疫组织化学染色结果均显示:与健康对照组相比,乙肝肝硬化患者单核细胞中Tim-3蛋白表达水平显著降低(p<0.01);但进一步对不同单核细胞亚群进行分析发现,CD14+CD16-单核细胞亚群中Tim-3蛋白的表达水平也显著下降(p<0.01),而CD14+CD16+单核细胞亚群中Tim-3蛋白的表达水平则无显著差异(P =0.5732)。结论:本研究结果证实Tim-3蛋白在乙肝肝硬化患者和健康对照组人群外周血单核细胞中存在表达差异,而且在CD14+CD16-/CD14+CD16+亚群单核细胞中表达也存在差异,提示Tim-3蛋白可能参与了乙肝肝硬化患者单核细胞介导的免疫或炎症反应,其具体机制需要进一步研究。第二部分LPS通过抑制Tim-3蛋白促进乙肝肝硬化患者单核细胞炎症因子分泌的研究研究目的:通过检测并分析乙肝肝硬化患者和健康对照人群外周血中LPS及单核细胞Tim-3蛋白表达相关程度,并鉴定单核细胞在LPS刺激下多种炎症因子的分泌水平以及不同亚群单核细胞表面TLR4表达差异,从而揭示LPS在乙肝肝硬化患者体内是如何通过调控单核细胞内的Tim-3蛋白而影响炎症反应过程。研究方法:采集受试者外周血样本并通过:①基质染色法鲎试剂盒检测血清中LPS含量,比较乙肝肝硬化患者与健康对照组人群LPS水平差异;②流式细胞仪检测Tim-3蛋白表达,并与LPS水平进行相关性分析;③流式细胞仪分选单核细胞亚群,通过体外培养并给予LPS刺激,ELISA试剂盒检测培养液中TLR4以及TNF-αα、IL-6、IL-1β、NF-κκB等炎症分子的表达水平。结果:与对照组相比,乙肝肝硬化患者血清中LPS水平显著升高(p<0.001),且LPS水平与单核细胞中Tim-3蛋白的表达水平呈负相关(r=-0.5287 P<0.0001)。ELISA检测结果显示,TLR4在乙肝肝硬化患者的单核细胞以及CD14+/CD16-亚型单核细胞中表达均显著升高(p<0.01);且在LPS刺激以后乙肝肝硬化患者血清中TNF-α IL-6、IL-1β、NF-κB炎症因子表达明显升高(p<0.01)。结论:本研究结果提示乙肝肝硬化患者外周血单核细胞Tim-3蛋白表达量下降与LPS有关,Tim-3参与LPS诱发的CD14+单核细胞炎症反应,体外LPS刺激可能通过单核细胞表面TLR4介导促进TNF-α IL-6、IL-1β、NF-κκB等多种炎症因子分泌,从而影响乙肝肝硬化炎症反应进程。第三部分microRNA-545在转录水平下调Tim-3基因表达的研究研究目的:通过在线软件数据库预测参与调控Tim-3基因表达的可能非编码RNA,并进一步通过双荧光素酶报告基因证实具体调控位点,从而揭示Tim-3在乙肝肝硬化患者体内表达失调的基因转录调控机制。研究方法:利用在线软件数据库microRNA database(www.mirdb.org)预测调控Tim-3基因 mRNA 的 3’UTR 区域的microRNA(microRNA-545):①Real-time PCR检测乙肝肝硬化患者以及健康对照组人群外周血单核细胞中的microRNA-545、1ncRNA Ftx及Tim-3的mRNA表达水平;②将预测得到的靶位点序列或其突变序列克隆到双荧光素酶报告基因载体中,然后转染293T细胞进行靶位点检测;从而验证miR-545对Tim-3基因上3’UTR区靶点的直接调控作用。结果:根据软件预测获得microRNA-545直接靶向Tim-3基因3’UTR区域,Real-time PCR结果显示:与健康对照人群相比,microRNA-545在乙肝肝硬化患者外周血单核细胞中高表达(p<0.01),而1ncRNA Ftx和Tim-3 RNA呈现低表达(p<0.01)。双荧光素酶基因系统正向检验:与对照组相比,过表达microRNA-545能显著下调Tim-3基因的表达(p<0.01)。突变序列报告基因系统,对Tim-3基因的3’ UTR区域突变后,与对照组相比,过表达microRNA-545则对荧光素酶活性没有显著差异;而降低内源的microRNA-545的水平能够提高荧光素酶活性。结论:本研究结果证实microRNA-545直接负调控Tim-3基因mRNA表达,这种调控作用是通过与Tim-3基因的3’UTR区直接结合发挥作用,而且可能与1ncRNA Ftx表达下调有关。由此,我们初步证实非编码RNA(GncRNA Ftx/miR-545)在转录水平参与调控Tim-3基因在乙肝肝硬化患者外周血单核细胞中的表达。第四部分非编码RNA Ftx/microRNA-545通过下调Tim-3促进单核细胞炎症因子分泌的研究研究目的:通过在单核细胞中过表达或干扰1ncRNA Ftx以后,检测microRNA-545、Tim-3以及各种炎症因子分泌水平,从而判断1ncRNA Ftx/microRNA-545在乙肝肝硬化患者中对Tim-3表达以及炎症反应过程的调控作用。研究方法:通过分别构建1ncRNA Ftx的过表达载体或siRNA基因干扰载体,并转染单核细胞THP-1,进行体外培养:①收集细胞,提取总RNA,Real-time PCR检测microRNA-545和Tim-3 mRNA水平;②收集细胞并提取蛋白,通过Western-blot检测Tim-3蛋白表达水平;③收集培养液,通过ELISA试剂盒检测其中TNF-α、IL-6、IL-1β和NF-κB等炎症因子分泌水平。结果:与对照组相比,过表达1ncRNA Ftx能够显著降低单核细胞内microRNA-545的水平(p<0.001),同时,培养液中TNF-α、IL-6、IL-1β和NF-κκB等炎症因子的浓度也显著降低(p<0.001),Tim-3基因表达的mRNA和蛋白水平则均显著升高(p<0.001);而干扰1ncRNA Ftx以后则与上述结果相反。结论:本研究结果证实了 1ncRNA Ftx通过负调控microRNA-545的表达,介导对Tim-3基因转录的调控作用,并影响单核细胞对炎症细胞因子的分泌功能,从而证实了 1ncRNA Ftx/microRNA-545通过调控Tim-3参与乙肝肝硬化炎症反应进程。更多还原

【Abstract】 Cirrhosis caused by hepatitis B virus(HBV)infection is one of the major diseases that threaten the health of all human beings.There are about 2 billion people with HBV infection in the world,about 15 to 25%of patients in China die from diseases associated with HBV infection.It is believed that there are multiple stages of immune dysfunction in the development of hepatitis B virus related-cirrhosis(HBV-related cirrhosis).In the early stage,interventions can be taken in time,and the process of liver fibrosis can be reversed.Current research believes that the molecular mechanisms of acquired and innate immune function changes in different stages of HBV infection are different.Chronic hepatitis B(CHB)is mainly caused by virus-specific adaptive immune function,while chronic type B Hepatitis cirrhosis can cause disorders of innate immune function.Exploring the regulation mechanism of innate immune disorder in hepatitis B cirrhosis and its regulatory targets are of great significance for the research and development of new technologies and clinical new drugs for blocking disease progression.T-cell immunoglobulin and mucin domain-containing protein 3(Tim-3)was originally discovered and expressed as an auxiliary type I T lymphocyte(Help T cell 1,Th1).The specific surface molecules can be expressed in innate immune cells such as monocytes,dendritic cells,mast cells,etc.,and participate in the process of chronic hepatitis B cirrhosis,which has become a hot spot in chronic hepatitis B,cirrhosis and even liver cancer in recent years.In addition,studies have shown that CD14+CD16+monocyte subsets are significantly elevated in peripheral blood and liver tissues of patients with chronic hepatitis B.By secreting a large number of pro-inflammatory factors,liver inflammation persists and can directly activate stellate cells.It plays an important role in the formation of liver fibrosis and cirrhosis.At present,the expression of Tim-3 in monocyte subsets and whether it is involved in HBV infection-mediated hepatitis B cirrhosis remains to be further studied.The Tim-3 molecule is a type of transmembrane protein formed by transcription of the Tim gene family,which regulates the level of transcription and post-transcriptional modification,translation and post-translational modification.Non-coding RNA(ncRNA)is a new type of small RNA that has been newly discovered in recent years.It mainly participates in the regulation of transcription and translation of target genes by binding to target gene sequences,including microRNA(miRNA)and Long noncoding RNA.(1ncRNA),microRNA interacts with IncRNA to further regulate gene protein function by regulating target gene protein expression.In this paper,the expression level of Tim-3 in different types of mononuclear cell subsets in peripheral blood of HBV-related cirrhosis patients was detected,and it was found that Tim-3 was indeed misexpressed to varying degrees,which also affected the levels of various inflammatory and immune molecules in HBV-related cirrhosis patients.Further,we explored the causes of the malexpression of Tim-3 in chronic hepatitis b from the perspective of non-coding RNA,thus discovering and confirming that IncRNA Ftx and microRNA-545 are involved in the regulation of Tim-3 expression,an important regulatory pathway.This pathway may play an important role in the inflammatory process of hepatitis B cirrhosis.This topic is studied from the following four aspects:Part Ⅰ:Expression of Tim-3 protein in peripheral blood mononuclear cells of patients with hepatitis B cirrhosisObjective:By comparing the differential expression levels of Tim-3 protein in different peripheral blood cell subsets of patients with hepatitis B cirrhosis and healthy controls,Tim-3 protein was initially judged to play a corresponding role in the development of hepatitis B cirrhosis.Methods:Thirty-two HBV-related cirrhosis patients(experimental group)and Thirty-six healthy volunteers(control group)were collected for fresh peripheral anticoagulation.Mononuclear cells were isolated and paraffin blocks were made.Immunohistochemical staining was performed after sections to detect the protein expression of Tim-3.Results:Flow cytometry showed that Tim-3 expression level(including Tim-3 MIF)was decreased in CD14+ mononuclear subgroup of CHB cirrhosis patients compared with normal healthy people(p<0.01).At the same time,the expression level of Tim-3 in CD14+CD16-monocyte subgroup in HBV-related cirrhosis patients was also significantly decreased(p<0.01),while the level of Tim-3 in CD14+CD16+ monocyte subgroup was not significantly different from that in normal healthy control group(P=0.5732).Immunohistochemical staining results also showed that Tim-3 expression was significantly down-regulated in HBV-related cirrhosis peripheral blood monocytes.Conclusion:The results confirmed that Tim-3 in CHB patients with cirrhosis and normal healthy people express differences exist in the peripheral blood mononuclear cells in the body,at the same time it also exists in different monocyte subsets expression differences,suggesting that Tim-3 may participate in the development process of HBV-related cirrhosis,function and its specific mechanism deserves further exploration.Part Ⅱ:LPS promotes secretion of inflammatory cytokines in monocytes from patients with hepatitis B cirrhosis by inhibiting Tim-3 proteinObjective:Endotoxin(LPS)levels in peripheral blood serum of normal healthy people and CHB cirrhosis patients and their correlation with Tim-3 were detected.We also identified the secretion levels of various inflammatory factors and the expression of TLR4 on the surface of monocytes in different subpopulations of monocytes under LPS stimulation.This reveals how LPS affects the inflammatory process by regulating Tim-3 in monocytes in patients with hepatitis B cirrhosis.Methods:Peripheral blood samples of the subjects were collected:1.(2)flow cytometry was used to detect Tim-3 level,and the correlation between Tim-3 level and endotoxin level was analyzed.(3)isolate mononuclear cells from peripheral blood of HBV-related cirrhosis patients and healthy control group and add endotoxin stimulation.ELISA kit was used to detect the expression levels of inflammatory molecules such as TLR4,TNF-alpha,IL-6,IL-1 beta and NF-kappa B in the culture solution.Results:Compared with the control group,the serum LPS level was significantly increased in patients with hepatitis B cirrhosis(p<0.001),and the LPS level was negatively correlated with the expression level of Tim-3 protein in monocytes(r=-0.5287 P<0.0001).In addition,TLR4 was highly expressed in the total mononuclear cells isolated from patients with HBV-related cirrhosis by ELISA(p<0.01).Furthermore.ELISA experiments showed that TNF-alpha,IL-6,IL-1 beta and NF-kappa B increased significantly in HBV-related cirrhosis patients after LPS stimulation(p<0.01).Conclusion:The results of this study suggest that the expression of Tim-3 protein in peripheral blood mononuclear cells of patients with hepatitis B cirrhosis is related to LPS,and Tim-3 is involved in LPS-induced CD14 monocyte inflammatory response.In vitro LPS stimulation may promote the secretion of inflammatory factors such as TNF-α,IL-6,IL-1β and NF-κB through the TLR4 on the surface of monocytes,thus affecting the inflammatory process of cirrhosis.Part III:MicroRNA-545 down-regulates Tim-3 gene expression at the transcript tional levelObjective:To identify and verify the non-coding RNA molecules involved in the regulation of Tim-3 expression,so as to reveal the causes of expression imbalance of Tim-3 in patients with HBV-related cirrhosisMethods:The microRNA database(www.mirdb.org),an online software database,was used to predict the microRNA in the 3 ’UTR region that regulates the mRNA of Tim-3 gene.(1)Real-time PCR detection of mRNA expression of microRNA-545,1ncRNA Ftx and Tim-3 in healthy controls and mononuclear cells of patients with hepatitis B cirrhosis(2)the predicted target point sequence or its mutant sequence was cloned into the dual luciferase reporter gene vector,and then transfected into 293T cells for target point detection;The direct regulatory effect of miRNA on target sites in the 3’UTR region of Tim-3 was verified from both positive and negative aspects.Results:The software predicted that microRNA-545 directly targeted the 3 ’UTR region of Tim-3 gene.Real-time PCR results showed that microRNA-545 was highly expressed in monocytes of CHB cirrhosis patients(p<0.01),which was contrary to the results of Tim-3.In addition,it was also found that 1ncRNA Ftx was poorly expressed in patients(p<0.01).Double-luciferase gene system positive test:compared with the control group,overexpression of microRNA-545 significantly down-regulated the expression of Tim-3 gene.In the mutant sequence reporter gene system,the over-expression of microRNA-545 showed no significant difference in luciferase activity compared with the control group after the mutation of the 3 ’UTR region of Tim-3 gene.In addition,reducing the level of endogenous microRNA-545 can improve luciferase activity.Conclusion:The results of this study confirmed that microRNA-545 directly negativelyregulates the mRNA level of Tim-3,which is achieved through direct binding of target sites in the 3 ’UTR region,and the expression of microRNA-545 is related to IncRNA Ftx,which initially revealed that non-coding RNA may be the cause of maladjustment of Tim-3 expression in mononuclear cells of HBV-related cirrhosis patients.Part Ⅳ:Non-coding RNA Ftx/microRNA-545 promotes monocytic inflammatory factor secretion by down-regulating Tim-3Objective:By over-expressing or interfering with lncRNA Ftx in monocytes,microRNA-545,Tim-3,and various inflammatory cytokine levels were examined to determine the expression of Tim-3 in IncRNA Ftx/microRNA-545 in patients with hepatitis B cirrhosis and The regulation of the inflammatory response process.Methods:LncRNA Ftx overexpression vector or siRNA gene interference vector were constructed and transfected into mononuclear cells for in vitro culture.(1)total RNA was collected and extracted.Real-time PCR was performed to detect RNA levels of microRNA-545 and Tim-3.(2)cells were collected,proteins were extracted,and Tim-3 protein levels were detected by western-blot.(3)the culture medium was collected and the concentrations of inflammatory factors such as TNF-alpha,IL-6,IL-1 beta and NF-kappa B were detected by ELISA.Results:Compared with the control group,the levels of endogenous microRNA-545 were significantly decreased by overexpression of IncRNA Ftx(p<0.001).At the same time,the concentrations of inflammatory factors such as TNF-alpha,IL-6,IL-1 beta and NF-kappa B were also significantly decreased(p<0.001).Meanwhile,the levels of mRNA and protein at Tim-3 were also increased(p<0.001).Interference with IncRNA Ftx was contrary to the above results.Conclusion:The results of this study confirmed that IncRNA Ftx negatively regulates the expression of microRNA-545,and then mediates the regulation of Tim-3.This regulation also affects the secretion of immune factors by monocytes,thus confirming that lncRNA Ftx/microRNA-545 participates in the inflammatory process of hepatitis B cirrhosis by regulating Tim-3.更多还原