【作者】 李翔；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 免疫学， 2019， 硕士

【摘要】 恒定自然杀伤T(invariant natural killer T,iNKT)细胞是同时具有NK(natural killer,NK)细胞和T细胞特征的独特细胞亚群,具有CD1d限制性,活化后能够分泌大量细胞因子,具有重要的免疫调节作用,是连接固有免疫和适应性免疫之间的桥梁。现有研究表明,iNKT细胞在人体肝脏中相对富集,可调控乙型肝炎病毒(Hepatitis B virus,HBV)感染后肝内免疫微环境,从而影响HBV感染后疾病的转归。然而,现有研究对于iNKT细胞在HBV感染中的细胞数量与细胞因子分泌功能变化出现过相互矛盾的报道,人体iNKT细胞在HBV感染过程中的效应尚不明确。本研究通过对不同临床分型的慢性HBV感染患者iNKT细胞数量与细胞因子分泌功能的meta分析,结合应用体外混合细胞培养模型,分析人iNKT细胞在HBV感染与非感染细胞刺激状态下的细胞因子分泌差异,并通过RNA-seq分析及公共数据集分析探讨基于感染细胞转录组相关机制。论文共分为两部分,其主要内容与结果如下:一、不同临床分型慢性HBV感染患者iNKT数量与细胞因子分泌功能变化的meta分析:系统搜索PubMed,Embase,Cochrane,Cnki和WanfangData等数据库,选择包含慢性HBV感染患者外周血或肝组织iNKT数量与细胞因子分泌功能变化的研究,进行meta分析。结果显示,外周血iNKT占CD3~+T的比例在不同临床分型的慢性HBV感染患者中呈现动态变化。在慢性HBV携带者(多为免疫耐受患者)中升高,其统计学指标为:与健康对照相比均值差(mean difference,MD)为0.16%,95%置信区间(confidential interval,CI)为[0.10%,0.23%],I~2=0%,p<0.00001。在慢性乙型肝炎患者(包括免疫清除期患者)、非活动性HBsAg携带者(又称非活动性携带者)和乙型肝炎肝硬化患者中均有不同程度的下降(与健康对照相比,慢性乙型肝炎患者MD=-0.10%,95%CI[-0.15%,-0.06%],I~2=42%,p<0.00001;慢性乙肝患者中免疫清除期患者MD=-0.14%,95%CI[-0.20%,-0.09%],I~2=0%,p<0.00001;非活动性HBsAg携带者MD=-0.05%,95%CI[-0.09%,-0.02%],I~2=0%,p=0.004;乙型肝炎肝硬化患者MD=-0.07%,95%CI[-0.09%,-0.04%],I~2=0%,p<0.00001)。对不同临床类型患者进行细胞因子分泌能力分析,结果显示:IFN-γ阳性表达的iNKT(IFN-γ~+iNKT)细胞比例在免疫清除期患者中升高(MD=9.40%,95%CI[3.33%,15.46%],I~2=0%,p=0.002),IL-4阳性表达的iNKT(IL-4~+iNKT)细胞比例在乙型肝炎肝硬化患者中升高(MD=8.88%,95%CI[3.94%,13.82%],I~2=0%,p=0.0004),提示IFN-γ~+iNKT细胞促进抗病毒免疫应答,IL-4~+iNKT细胞促进肝硬化的发生。二、HBV表达阳性与阴性肝癌细胞系刺激iNKT细胞分泌细胞因子的差异及其转录组学分析与公共数据挖掘:选择iNKT细胞系(转染了iNKT TCR的Jurkat CD8~+细胞株,hiNKT38)和两种表达CD1d的肝癌细胞系(HepG2-tmCD1d细胞、HepG2.2.15-tmCD1d细胞,后者表达HBV)分别作为效应细胞与刺激细胞,共培养24 h后检测培养上清中细胞因子的浓度。结果显示模拟HBV感染的HepG2.2.15-tmCD1d刺激组与对照HepG2-tmCD1d刺激组相比,hiNKT38细胞IFN-γ分泌显著减少。将刺激细胞样本送交转录组测序,结果提示HepG2.2.15-tmCD1d细胞IL-18、IL-12A表达下调。另外,对GEO数据库(高通量基因表达数据库)的两个数据集分析也显示感染HBV的人原代肝细胞的IL-18编码基因下调表达。这些结果提示HBV感染可抑制肝细胞内IL-18、IL-12A的表达量,并导致iNKT细胞IFN-γ分泌减少。本研究的结论和意义:1.慢性HBV感染患者外周血iNKT细胞数量与细胞因子分泌功能变化与临床分型相关,IFN-γ~+iNKT细胞比例和IL-4~+iNKT细胞比例分别与抗病毒免疫应答和肝硬化的发生相关。2.人iNKT细胞系hiNKT38对模拟HBV感染的HepG2.2.15-tmCD1d细胞表现出IFN-γ分泌减少。转录组分析结果提示HBV抑制HepG2.2.15-tmCD1d细胞IL-18、IL-12A基因表达量。来源于GEO数据库(高通量基因表达数据库)的两个数据集的结果也显示感染HBV的人原代肝细胞的IL-18编码基因下调表达。本研究的创新点:本论文对慢性HBV感染中iNKT的数量和功能进行meta分析,通过细胞实验分析hiNKT38对于HBV表达阴阳性的两种肝癌细胞系的差异性细胞因子分泌,研究了在HBV刺激下HepG2.2.15-tmCD1d的差异表达基因,结果提示HBV可能通过抑制HepG2.2.15-tmCD1d细胞IL-18、IL-12A的表达逃避iNKT的免疫攻击。更多还原

【Abstract】 Invariant NKT(iNKT)cells are unique cell subsets with the characteristics of both NK cells and T cells.They are CD1d-restricted and can secrete a large number of cytokines after activation.They have important immunomodulatory effects and are a bridge between innate immunity and adaptive immunity.Existing studies have shown that iNKT cells are abundantly enriched in the human liver and can regulate the intrahepatic immune microenvironment after HBV infection,which can affect the outcome of diseases after HBV infection.However,there have been conflicting reports on the changes of number and cytokine secretion function of iNKT cells in HBV infection.The effects of human iNKT cells in the process of HBV infection is still unclear.In this study,the meta-analysis of the number and cytokine secretion function of iNKT cells in patients with chronic HBV infection in different disease statuses was perpromed.To further verify the effect of HBV on iNKT cell cytokine producing capapcity,iNKT cell clone hiNKT38 was stimulated with CD1d-bearding HBV-infected(HepG2.2.15-tmCD1d)and non-infected(HepG2-tmCD1d)cells,followed by cytokine detection.In addition,stimulator-based mechanisms was explored by RNA-seq analysis and public dataset analysis.The paper is divided into two parts,the main contents and results are as follows.1.Meta-analysis of the number and cytokine secretion function of iNKT in patientswith chronic HBV infection in different clinical typesAfter systematically searching for studies about the changes in the number and cytokine secretion function of iNKT in peripheral blood or liver tissues of patients with chronic HBV infection on PubMed,Embase,Cochrane,Cnki and WanfangData,the results of meta-analysis showed that the ratio of iNKT to CD3~+T in peripheral blood changes dynamically in different clinical types of the disease.Compared to healthy controls,it increased in chronic HBV carrier,whose statistical indicator comprise that MD(mean difference)is 0.16%,95%CI is[0.10%,0.23%],I~2 is 0%,and p value is less than 0.00001.It has varying degrees of decline in patients with CHB(chronic hepatitis B),CHB patients in immune clearance phase,inactive HBsAg carriers(also known as inactive carriers),and patients with HBV-LC(HBV-liver cirrhosis).The statistical indicator of CHB patients comprise that MD is-0.10%,95%CI is[-0.15%,-0.06%],I~2is 42%,and p value is less than 0.00001.The statistical indicator of CHB patients in immune clearance phase comprise that MD is-0.14%,95%CI is[-0.20%,-0.09%],I~2is 0%,and p value is less than 0.00001.The statistical indicator of inactive HBsAg carriers comprise that MD is-0.05%,95%CI is[-0.09%,-0.02%],I~2 is 0%,and p value is 0.004.The statistical indicator of patients with HBV-LC comprise that MD is-0.07%,95%CI is[-0.09%,-0.04%],I~2 is 0%,and p value is less than 0.00001.Analysis of cytokine secretion ability in patients with different clinical types showed that the ratio of IFN-γ~+iNKT increased in CHB patients in immune clearance phase,whose statistical indicator comprise that MD is 9.40%,95%CI is[3.33%,15.46%],I~2 is 0%,and p value is 0.002.The ratio of IL-4~+iNKT increased in patients with HBV-LC,whose statistical indicator comprise that MD is 8.88%,95%CI is[3.94%,13.82%],I~2 is 0%,and p value is 0.0004.The results suggested that IFN-γ+iNKT cells promoted antiviral immune response and IL-4~+iNKT cells promoted the development of cirrhosis.2.Differential cytokine secretion by iNKT cells stimulated by two kinds of humanhepatoma cell lines positive or negative for HBV expression and transcriptomicanalysis of hepatoma cells and public data miningThe iNKT cell line derived from Jurkat CD8~+cells transfected with TCR of iNKT(hiNKT38)and two hepatoma cell lines expressing CD1d(HepG2-tmCD1d cells,HepG2.2.15-tmCD1d cells expressing HBV)were selected as effector cells and stimulating cells,respectively.The concentration of cytokines in the culture supernatant was determined through CBA after 24 hours of co-culture.The results showed that the IFN-γsecretion of hiNKT38 cells was significantly decreased in the HepG2.2.15-tmCD1d stimulation group imitating HBV infection compared with the HepG2-tmCD1d stimulation group.The stimulating cell samples were sent for the transcriptome sequencing.The results of RNA-seq analysis indicated that the expression of IL-18 and IL-12A was declined in HepG2.2.15-tmCD1d cells.In addition,the results of two datasets from the GEO database(high-throughput gene expression database)also showed down-regulated expression of the IL-18-encoding gene in human primary hepatocytes infected with HBV.These results suggested that HBV can reduce the IFN-γsecretion of iNKT cells by inhibiting IL-18 and IL-12A expression in hepatocytes.The conclusions and significance of this study1.The number and cytokine secretion function of iNKT cells in peripheral blood ofpatients with chronic HBV infection is related to clinical types.The proportion ofIFN-γ+iNKT cells and IL-4~+iNKT cells were related to antiviral immune responseand the occurrence of cirrhosis respectively.2.The human iNKT cell line hiNKT38 showed a decrease in IFN-γsecretion underthe stimulation of HepG2.2.15-tmCD1d cells imitating HBV infection.Transcriptome analysis indicated that HBV inhibited IL-18 and IL-12A geneexpression in HepG2.2.15-tmCD1d cells.Results of two datasets from the GEOdatabase(high-throughput gene expression database)also showed down-regulatedexpression of the IL-18-encoding gene in human primary hepatocytes infected withHBV.The innovation of this researchA meta-analysis of quantity and function of iNKT in chronic HBV infection was performed.The differential cytokine secretion of hiNKT38 in the presence of two hepatoma cell lines positive or negative for HBV expression was analyzed through cell experiment.The differentially expressed genes of HepG2.2.15-tmCD1d under HBV stimulation were studied.The results suggested that HBV may evade immune attack of iNKT cells by inhibiting IL-18 and IL-12A expression in HepG2.2.15-tmCD1d cells.更多还原