【作者】 陈韬；

【导师】 宁琴；

【作者基本信息】 华中科技大学 ， 免疫学， 2010， 博士

【摘要】 [研究背景及目的]我国是HBV感染的高发地区,1-59岁人群携带率约7.18%,由HBV感染导致的重型肝炎或肝功能衰竭仍然是我国临床常见急危重疾病。我国学者将肝功能衰竭的临床分型定为：急性肝衰竭(Acute Liver Failure or Fulminant Hepatits, FHF)、亚急性肝衰竭(Subacute Liver Failure, SALF)、慢加急性肝衰竭(Acute-on-Chronic Liver Failure, ACLF)和慢性肝衰竭(Chronic Liver Failure, CLF),其病情凶险,特别是急性肝衰竭死亡率高达70%-90%,临床上缺乏特异、有效的治疗手段和干预靶点,除非紧急实施肝移植(国外以肝移植为主要治疗手段,肝移植后5年存活率约为50%～60%),大部分患者预后不良,并发症多。病毒性重型肝炎的发病机制十分复杂,一般认为是病毒和宿主的相互作用的结果,现今较为公认的是“两次损伤学说”,即由病毒直接或间接(免疫反应)所致的原发性损伤和内毒素—细胞因子轴—肝损伤学说为核心的继发性损伤,加之患者肝脏储备功能较差更易导致乙型肝炎重症化。在国外,重型肝炎多由药物性因素引起,而在我国则多由HBV感染所致。由于HBV病毒是一种非溶细胞性嗜肝病毒,感染后造成肝损伤的主要机制是机体的免疫系统主动清除被感染的肝细胞,因而对HBV感染后机体免疫功能的研究对于阐述乙型肝炎的发病和进展等方面的机制具有针对性的作用。在本实验室的前期工作中,我们利用人全基因组芯片技术(8600个探针),分析了重型乙型肝炎患者和轻中度慢性乙型肝炎患者外周血淋巴细胞(PBMC)的基因差异表达谱,筛选出了在重型肝炎中高表达的263种基因,涉及多个类别。KCTD9 (Potassium Channel Tetramerisation Domain Containning 9)和KCNJ15 (Potassium Inwardly-rectifying Channel, Subfamily J, Member 15)是其中的两种与离子通道相关的基因,在重型肝炎中这两种基因表达分别上调6倍和7倍,通过后续的扩大样本的验证实验证实了该结果的可靠性。进一步从基因和蛋白水平,我们检测了265例临床不同类型HBV感染患者包括轻中度和重型乙型肝炎患者、以及健康对照人群的肝组织和PBMC中KCTD9分子的表达水平,发现其仅在重型乙型肝炎患者有显著高表达,提示其与病毒性乙型肝炎疾病严重程度的相关性。为深入探讨KCTD9分子在重型乙型肝炎发生发展中的作用及可能的机制,我们利用本实验室建立的MHV-3诱导的Balb/cJ小鼠暴发型肝炎模型开展了研究,发现KCTD9分子在MHV-3病毒感染后的小鼠肝组织和肝脏淋巴细胞尤其是NK细胞中高度表达,与前期人类重型乙型肝炎的研究结果相似,提示可利用该模型深入剖析KCTD9参与HBV病毒诱导的重型肝炎肝细胞损伤的发生发展机制。我们的前期工作首次表明,在病毒诱导的急性肝衰竭中肝脏NK细胞通过Fas/FasL和NKG2D/NKG2DL途径增强对肝细胞的杀伤,本研究的主要目标是进一步阐明肝脏NK细胞在急性肝衰竭中的作用及作用机制,研究NK细胞高表达KCTD9分子在肝细胞损伤和重型肝炎发生发展过程的作用及作用机制。具体研究内容如下：一、NK细胞在重型病毒性肝炎中的作用及其免疫学机制研究1、在HBV-ACLF患者肝组织中检测NK细胞的分布,同时在外周血NK细胞中检测FasL、NKP30和NKP46的表达,探讨NK细胞参与重型乙型肝炎急性肝损伤的机制；2、在MHV-3诱导的Balb/cJ小鼠暴发型肝炎模型中,体内耗竭NK细胞,观察干预效果,探讨NK细胞参与急性肝损伤的作用。二、NK细胞新型分子KCTD9在重型乙型肝炎中的作用及其免疫学机制研究1、以重型乙型肝炎患者为对象的研究(1)收集重型乙型肝炎患者和轻中度慢性乙型肝炎患者外周血PBMC,对表达KCTD9的淋巴细胞亚群进行定位。(2)分析表达KCTD9分子的外周血NK细胞与重型乙型肝炎肝损伤严重程度的相关性。2、利用人NK92细胞系进行KCTD9分子的体外功能研究。3、以MHV-3诱导的Balb/cJ小鼠暴发型肝炎模型为对象的研究。(1)构建针对小鼠KCTD9的shRNA干扰质粒,非相关干扰质粒以及小鼠KCTD9的全长表达质粒,观察在小鼠暴发型肝炎模型中干扰或过表达KCTD9对于疾病病程的影响。(2)在小鼠暴发型肝炎模型中观察干扰或过表达KCTD9对于淋巴细胞功能的影响,研究KCTD9参与暴发型肝炎NK细胞介导的急性肝损伤的作用机制。[研究方法]1、利用免疫组织化学技术,检测HBV-ACLF患者和轻中度CHB患者肝组织中NK细胞的表达频数；利用流式细胞术分析HBV-ACLF患者和轻中度CHB患者外周NK细胞FasL、NKP30和NKP46的表达情况。利用尾静脉注射抗ASGM-1抗体耗竭NK细胞,观察MHV-3诱导的Balb/cJ小鼠暴发型肝炎模型的生存率；2、利用流式细胞术检测KCTD9分子在重型乙型肝炎患者和轻中度慢性乙型肝炎患者PBMC中NK细胞、CD4+T细胞和CD8+T细胞的表达水平,并收集病例资料,统计分析表达KCTD9的NK细胞比例与肝损伤严重程度的相关性。同时利用免疫组织化学技术检测重型乙型肝炎患者肝组织连续切片中NK细胞表达KCTD9的情况。3、利用NK92细胞系,将人KCTD9表达质粒电转染入该细胞,流式细胞术观察转染24小时后NK92细胞KCTD9蛋白和活化分子CD69的表达；将转染后的NK92细胞与HepG2.2.15细胞按照梯度效靶比共培养,通过测定ALT释放量,计算NK92细胞的杀伤效率；ELISA法检测转染后NK92细胞分泌IFN-γ和TNF-α的情况；利用流式细胞术筛选转染后NK92细胞的活化性／抑制性受体表达谱。4、构建针对小鼠KCTD9的shRNA干扰质粒和全长表达质粒,利用尾静脉高压注射技术,将KCTD9干扰质粒或表达质粒注射入MHV-3诱导的暴发型肝炎模型小鼠体内,从小鼠的生存情况、血清转氨酶水平、肝脏组织学改变等方面研究KCTD9分子在暴发型肝炎疾病病程中的作用。流式细胞术检测shRNA干扰对于本模型小鼠肝脏NK细胞活化和KCTD9表达的影响,利用磁珠分选技术将肝脏NK细胞分离纯化,体外检测其针对MHV-3感染的肝细胞的杀伤活性。流式细胞术检测shRNA干扰后肝脏NK细胞分泌的细胞因子(IFN-γ和TNF-α)水平。[实验结果]1、NK细胞在病毒诱导的急性肝衰竭肝损伤中发挥重要作用。免疫组织化学检测结果显示,HBV-ACLF患者肝组织中NK细胞的表达显著高于轻中度CHB患者；FasL在HBV-ACLF患者肝组织中的表达显著高于轻中度CHB患者；表达NKP30、NKP46和FasL的外周NK细胞比例以及MFI水平均高于轻中度CHB患者。在MHV-3诱导的Balb/cJ小鼠暴发型肝炎模型中,耗竭NK细胞可以使该模型动物生存率由0显著上升到22.2%。2、KCTD9在HBV-ACLF患者外周血淋巴细胞的表达增高,以NK细胞最为显著。HBV-ACLF患者(15例)外周血中,表达KCTD9分子的NK细胞和CD4+T细胞的比例显著高于轻中度CHB患者(21例),而CD8+T细胞的比例无显著差义；HBV-ACLF患者外周NK、CD4~+T和CD8+T细胞表达KCTD9的平均荧光强度(MFI)水平均显著高于轻中度CHB患者。将15例HBV-ACLF患者表达KCTD9的外周NK细胞比例与患者临床指标进行相关性分析发现,其与ALT和AST水平高度正相关,而与Tbil、Dbil、ALB、PTA、HBV DNA以及年龄无相关性,提示KCTD9可能参与了NK细胞介导的肝细胞损伤及重型肝炎的发生。3、KCTD9通过下调NKG2A表达在体外促进NK92细胞活化。利用人KCTD9表达质粒电转染NK92细胞系24小时后,NK92细胞表达KCTD9分子和活化标志CD69分子水平均显著增加；其在体外针对HepG2.2.15细胞的杀伤功能显著增强：IFN-γ分泌增加；对系列NK细胞活化性／抑制性受体检测后发现NKG2A的表达显著下调。4、针对KCTD9的治疗性shRNA干扰延缓MHV-3诱导的暴发型肝炎疾病进展。成功构建小鼠KCTD9表达质粒、针对小鼠KCTD9的shRNA干扰质粒和非相关对照干扰质粒。利用构建的干扰质粒尾静脉高压注射MHV-3感染的Balb/cJ小鼠,可以使小鼠肝脏KCTD9的表达显著下调；小鼠的生存率由0上升到22.2%：血清转氨酶水平显著下降；小鼠肝组织的病理情况得到改善。KCTD9表达质粒注射组小鼠疾病病情加重。5、针对KCTD9的治疗性shRNA干扰降低肝脏NK细胞活化和杀伤功能。与非相关对照组相比,MHV-3感染后72小时shRNA干扰组肝脏NK细胞表达KCTD9和CD69分子均显著下降；分离纯化的肝脏NK细胞对感染后48小时肝细胞的杀伤效率显著降低；肝脏NK细胞分泌IFN-γ和TNF-α显著下降。KCTD9表达质粒注射组小鼠肝脏NK细胞活化和各项功能均显著增强。[结论]1、本研究在人类HBV-ACLF疾病及小鼠暴发型肝炎模型中证实了NK细胞在肝脏组织中大量存在,活化的肝脏NK细胞可通过增强表达的FasL发挥杀伤功能,进而在病毒诱导的急性肝损伤中发挥重要作用。2、体外研究阐明了KCTD9分子通过降低NKG2A受体的表达促进NK细胞活化,从而诱导之后的杀伤及细胞因子分泌功能。3、本研究阐明了KCTD9通过活化NK细胞从而参与病毒诱导的急性肝细胞损伤及重型肝炎发生发展过程中。4、小鼠暴发型肝炎模型中,干预KCTD9的表达可以通过降低肝脏NK细胞的活化而有效延缓病情进展,提高生存率,为重型肝炎的诊断和治疗提供了新的分子靶点和理论依据。更多还原

【Abstract】 [BACKGROUND & OBJECTIVE]The prevalence of HBV infection is high in China, where HBV carriers account about 7.18%in the group of age 1-59 by 2010. HBV induced severe hepatitis or hepatic failure is still a common disease with acute and severe manifestation. According to the consensus from Chinese Association for the Study of Liver and Infectious Disease, the hepatic failure can be classified as Acute Liver Failure or Fulminant Hepatitis (ALF), Subacute Liver Failure (SALF), Acute-on-Chronic Liver Failure (ACLF), and Chronic Liver Failure (CLF). Virus induced hepatic failure, especially acute liver failure which can lead to a 70-90% death rate, usually presents a emergent clinical process and results in multiple complications. Due to lack of specific and effective treatment and interference targets, the clinical outcome of patients with liver failure is poor unless emergent liver transplantation is appliedThe pathogenesis of virus induced hepatic failure is complicated and not well defined. However, it is generally accepted as the result of complex interaction between the virus and the host. The virus directly or indirectly induces activation of immune system which results in primary liver injury, subsequently down stream liver injury is further led by endotoxin-cytokines axis. Patients with long term of chronic liver disease can easily undergo severe hepatitis upon the reactivation of virus or host immune system. As we know, hepatic failure is mainly caused by drug agents in Western, while mainly by HBV infection in China. Since HBV virus is a kind of non-cytopathic virus and does not cause damage of hepatocytes directly, the primary mechanism of hepatocytes injury after HBV infection lies on clearance of virus infected hepatocytes by active action of immune system. So, exploration of the immunological role and mechanism in virus infection may be specific in elaboration of pathogenesis of hepatitis B virus induced hepatic failure.In our previous work, human whole genomic gene chip was adopted to analyze the disparate gene expression in PBMC from patients with HBV-ACLF or mild chronic hepatitis B. Among the 8600 cDNA probes, widely-spread 263 genes were substantially up-regulated in HBV-ACLF group. KCTD9 and KCNJ15, two kinds of ion channel genes, were most prominent identified, the expression of which were up-regulated 6 and 7 times in PBMCs of HBV-ACLF patients respectively when compared to mild/moderate CHB patients. And the following studies from amplified cases of different clinical types with HBV infection confirmed the reliability. Furthermore, we detected KCTD9 expression in liver tissue or PBMC from 265 patients with mild/moderate CHB or HBV-ACLF and 36 healthy controls in gene and protein levels. The results showed the expression of KCTD9 was prominently increased only in liver tissue and PBMCs from HBV-ACLF patients, which suggested a close relationship between KCTD9 expression and severity of hepatitis B. To explore the role and mechanism of KCTD9 in pathogenesis of HBV induced hepatic failure, similar study was performed in the MHV-3 induced fulminant hepatitis model in Balb/cJ mice. The result showed that KCTD9 highly expressed in the liver tissue and hepatic lymphocytes after MHV-3 infection especially the hepatic NK cells. The results in the animal model study were similar with that in human disease, which suggested the applicability of this animal model in further research with regards to the identification of the function and mechanism of KCTD9 involved in virus induced hepatocytes injury.Our previous work first elaborated the increased killing of hepatic NK cells by Fas/FasL and NKG2D/NKG2DL contributed to hepatocyte necrosis in virus induced liver failure. The aim of this study wass to further elaborate the role and mechanism of NK cells involved with acute hepatocyte injury in virus induced liver failure, as well as to investigate the function and pathogenesis of increased expression of KCTD9 on NK cells in the disease. The concrete contents were as the followings:Part 1 The role and immune mechanism of hepatic NK cells in virus induced liver failure.1. To detect distribution of NK cells in liver tissue and expression of FasL, NKP30 and NKP46 in peripheral NK cells from HBV-ACLF patients, and to discuss possible mechanism of acute liver injury that NK cells involved in HBV induced hepatic failure.2. To investigate the effect by depletion of NK cells in MHV-3 induced fulminant hepatitis mice and discuss the role of NK cells in the model.Part 2 The role and immune mechanism of novel KCTD9 in virus induced liver failure.1. The cellular localization and role of KCTD9 expression in patients with HBV-ACLF.(1) To locate the subpopulation of the peripheral lymphocytes with increased KCTD9 expression in patients with HBV-ACLF.(2) To analyze the correlation between the percentage of peripheral NK cell that expressed KCTD9 and severity of liver injury in patients with HBV-ACLF.2. The functional study of KCTD9 in a NK cell line (NK92 cell line).3. Th role and functional study of KCTD9 in a MHV-3 induced fulminant hepatitis model in Balb/cJ mice.(1) Construction of specific shRNA interference plasmid targeting KCTD9, irrelevant plasmid, and full length mouse KCTD9 expression plasmid, and verification of their interference effects on KCTD9 expression in vitro and vivo, and mice suvival, hepatic biochemistry and histopathologic improvement in MHV-3 infected Balb/cJ mice.(2) Investigation of KCTD9 expression in NK lymphocytes and its contribution to NK cell functional change in MHV-3 infected Balb/cJ mice.[METHODS]1. The frequencies of NK cells in the liver tissue of patients with HBV-ACLF or mild/moderate CHB were analyzed by Immunohistochemistry. The expression levels of FasL, NKP30 and NKP46 in peripheral NK cells were detected by FACS. Survival rate after depletion of NK cells by injection of anti-AsGMl through tail vein was observed in the mice model of FHF in MHV-3 infected Balb/cJ mice. 2. The expression levels of KCTD9 in peripheral NK cells, CD4+T cells, and CD8+T cells from patients with HBV-ACLF or mild/moderate CHB were detected by FACS. The clinical information of these patients was documented and the correlation between the percentage of NK cells that expressed KCTD9 and severity of liver injury was analyzed in HBV-ACLF patients. Meanwhile, KCTD9 expression on hepatic NK cells in the consecutive section of liver tissue was observed by Immunohistochemistry.3. The human KCTD9 expression plasmid was transfected into NK92 cell line by electronic transfection in vitro, the expression level of KCTD9 protein and activation marker CD69 were detected by FACS at 24h after transfection. The transfected NK92 cells were co-cultured with HepG2.2.15 cells in gradients of effector/target ratio to determine cytotoxicity of NK92 cells by quantitative measurement of ALT release, whereas the secretion of IFN-γand TNF-αby NK92 cells was detected. The profiling of functional receptors of NK92 cells after transfection was also analyzed by FACS.4. The shRNA interference plasmid targeting mouse KCTD9 gene and KCTD9 full length expression plasmid were constructed and introduced into MHV-3 infected Balb/cJ mice respectively by hydrodynamic delivery through tail vein. The effect of KCTD9 on the process of the disease was elucidated by survival rate, serum ALT level, and change of liver histopathology. Meanwhile, the effects of shRNA interference on activation and KCTD9 expression of hepatic NK cells were also evaluated by FACS. Moreover, hepatic NK cells were isolated by microbeads after shRNA interference, and then the cytotoxicity to MHV-3 infected hepatocytes in vitro, production of IFN-γand TNF-αby intracellular staining were all analysed.[RESULTS]1. NK cells play a significant role in virus induced acute liver injury. By immunohistochemistry the frequency of NK cells in liver tissue of patients with HBV-ACLF was much higher than that in patients with mild/moderate CHB. The expression level of FasL in liver tissue, percentage and MFI of NKP30, NKP46 and FasL on peripheral NK cells from patients with HBV-ACLF were prominently higher than those in patients with mild/moderate CHB.In MHV-3 induced fulminant hepatitis model, depletion of NK cells led to a increase of mice survival from 0.00%to 22.2%.2. There was increased expression of KCTD9 in peripheral lymphocytes, especially in NK cells from patients with HBV-ACLF. The percentage of peripheral NK and CD4+T cells but not CD8+T cells with elevated KCTD9 expression in patients(15 in total) with HBV-ACLF were evidently higher than that in patients (21 in total) with mild hepatitis B. The mean fluorescence index (MFI) of KCTD9 in peripheral NK, CD4+T, and CD8+T cells were all significantly increased in patients with HBV-ACLF. There was a positive correlation between the percentage of peripheral NK cells with increased KCTD9 expression and patients ALT or AST level, but not correlation with TBIL, DBIL, ALB, PTA, HBV DNA level and age, suggesting KCTD9 expression may contribute to NK cell mediated liver injury in virus induced hepatic failure.3. KCTD9 promoted activation of NK92 cells in vitro by down-regulating NKG2A expression. A human KCTD9 full-length expression recombinant plasmid (pcDNA3.1-hKCTD9) was constructed and introduced into NK92 cells, significantly elevated KCTD9 protein and activation marker CD69 were observed at 24h after transfection. Meanwhile, enhanced cytotoxicity of NK92 cells to HepG2.2.15 and increased secretion of IFN-r were also observed. After a series analysis of the functional receptors on transfected NK92 cells, a decrease of NKG2A expression was found indicating the mechanisms for KCTD9 to promot NK cells cytocoxicity may be mediated by downregulated expression of an inhibitory molecule NKG2D.4. The therapeutic shRNA interference plasmid targeting KCTD9 ameliorated the progress of MHV-3 induced fulminant hepatitis. A full length mouse KCTD9 expression plasmid, a specific KCTD9 shRNA and an irrelevant shRNA interference plasmid were successfully constructed. Introduction of a specific KCTD9 shRNA interference plasmids by hydrodynamic delivery into MHV-3 infected Balb/cJ mice, decreased KCTD9 expression in liver was observed and the survival rate elevated from 0 to 22.2%. Moreover, an extraordinary declined ALT level and significant improvement of liver histopathology was also evidenced when compared with the mice without treatment or with control plasmid. In contrast, hydrodynamic delivery of KCTD9 expression plasmid into MHV-3 infected Balb/cJ mice significantly deteriated the disease course. 5. The therapeutic shRNA interference plasmid targeting KCTD9 reduced activation and cytotoxicity of hepatic NK cells in MHV-3 incued fulminant hepatitis model. The KCTD9 and the activation marker CD69 in hepatic NK cells were notably down-regulated at 72 hours after delivery of a KCTD9 shRNA interference plasmid. Accordingly, the cytotoxicity of the hepatic NK cells from these mice to hepatocytes was decreased. These hepatic NK cells also displayed a decrease in IFN-y and TNF-a secreation in comparison with no treatment group or control plasmid treated mice. In contrast, an enhanced activation and cytotoxicity of hepatic NK cells was observed in mice when teil vein delivery of KCTD9 expression plasmid.[CONCLUSION]1. For the first time, this study explored prominently increased expression of NK cells in the liver tissue of HBV-ACLF patients and confirmed that activated hepatic NK cells contributed to hepatocytes necrosis by enhanced expression of FasL. This study also firstly elaborated the contribution of hepatic NK cells in virus induced FHF in a mice model.2. For the first time, this study discovered that the novel KCTD9 gene could promote the activation of NK92 cells in vitro via down-regulating the expression of its inhibotory molecule NKG2A.3. For the first time, this study discoved that KCTD9 gene plays an important role in the pathogenesis of acute hepatocyte injury and progress of virus induced hepatic failure by promoting activation of NK cells.4. For the first time, this study demonstrated a shRNA interference plasmid specific targeting KCTD9 gene inhibited NK cell activation and amelioated disease process in MHV-3 induced FHF, which in turn may shed light on the therapeutic stratitegies for disease control.更多还原