双阴性T细胞在小鼠病毒性暴发型肝炎中的作用研究

The Primarily Function of CD4~-CD8~-double Negative T Cell in MHV-3 Induced Murine Fμlminant Hepatitis

作者: 专业:免疫学 导师:宁琴 年度:2009 学位:博士 

关键词
T细胞 NK细胞 病毒性肝炎 3型鼠肝炎病毒(MHV-3)

Keywords
T cell, NK cell, viral hepatitis, Murine hepatitis virus strain 3 (MHV-3)
        【背景及目的】调节性T细胞是机体维持自身免疫平衡的重要组成部分,在肿瘤、移植、自身免疫性疾病和感染性疾病方面都有重要作用。近年来不同的调节性细胞克隆被陆续报道,引起了研究者的重视。CD4~+CD25~+Tregs作为经典的调节性T细胞,可通过白介素10(IL-10)和转移生长因子β(TGF-β)发挥免疫抑制效应,已经被广泛的研究和应用。此外,γδ~+T细胞、自然杀伤T细胞(NKT)、CD69~+CD4~+CD25~-T细胞、Qa-1限制性CD8~+T细胞和CD8~+CD28~-T细胞亦在各种动物实验模型中被发现具有免疫调节作用并被归为调节性T细胞家族。近年来,在器官移植研究领域发现了一种新型调节性T细胞——双阴性T(DN T,CD3~+CD4~-CD8~-double-negative)细胞。该细胞能够抑制杀伤抗供者CD4~+T细胞,CD8~+T细胞和B细胞,从而延长移植物存活时间。在自身免疫性疾病和衣原体感染中亦发现DN T细胞可发挥类似负性调节作用。然而,DN T细胞在机体内并非一成不变的表现出免疫抑制效应。在利什曼原虫感染中,人们发现利什曼原虫感染者外周血的DNT不同亚亚群跟病情发展方向相关,αβTCR~+CD3~+CD4~-CD8~-可加剧炎症的发展过程,γδTCR~+CD3~+CD4~-CD8~-可减轻炎症反应。在系统性红斑狼疮中DNT细胞通过CD1C限制性方式识别并诱导CD1C~+B细胞产生抗自身抗体,加重病情。迄今,DN T细胞是否在病毒性肝炎中发挥作用尚不明确。病毒感染是重症肝炎的主要原因之一。据统计我国乙型肝炎病毒(HBV)感染率高达10%,其中1%~4%患者最终进展为重症肝炎。该疾病以大面积肝细胞坏死、严重肝功能不良伴随肝性脑病为主要特征,可导致多器官功能衰竭,临床缺乏特异有效的治疗手段,如不及时实施肝移植,死亡率高达80%。重型乙型肝炎发病机制十分复杂,目前普遍认为病毒持续复制和宿主免疫防御的复杂相互作用导致了肝脏损伤,但是疾病演变过程中特异性细胞免疫调节机理至今不明。本实验室以鼠3型肝炎病毒(MHV-3,murine hepatitis virus 3)感染敏感系Balc/cJ小鼠成功建立了小鼠病毒性暴发型肝炎模型,本研究拟利用MHV-3诱导的小鼠暴发型肝炎模型,探讨DN T细胞在暴发型肝炎发生发展中的作用。具体研究目标如下:1.动态观察DN T细胞在病毒性暴发型肝炎小鼠体内的变化并分析其与疾病严重程度的关系。2.鉴定DN T细胞的表面标志和细胞因子谱。3.探讨DN T细胞在小鼠病毒性暴发型肝炎中的作用环节及可能机制。【方法】1.MHV-3腹腔感染途径建立小鼠暴发型肝炎模型,伊红-苏木精染色观察肝脏组织病理学变化,检测感染后不同时间点血清ALT,AST,TBiL水平,观察不同时间点小鼠肝脏、脾脏和外周血的DNT细胞比例变化。2.流式细胞术绝对计数观察DN T细胞在肝脏、脾脏和外周血等器官的数量变化,体外趋化实验观察MHV-3感染肝脏细胞对脾脏淋巴细胞亚群的趋化作用,探讨DN T细胞在暴发型肝炎小鼠病程中的迁移——再分布规律。3.流式细胞术检测DN T细胞的表面标志包括TCRβ、TCRγδ、CD30、CD25、CD44、CD28和细胞因子谱包括INF-γ、IL-2、IL-4、IL-10、IL-13、IL-17、TNF-α,合成α-Galcer二聚体来鉴定DN T细胞与NKT细胞之间的关系。流式细胞术检测4.磁珠分选DN T细胞、CD8~+T细胞、CD4~+T细胞和NK细胞。胶原酶原位灌流消化法分离小鼠原代肝细胞。乳酸脱氢酶(LDH,Lactate dehydrogenase)释放实验检测DN T细胞在体外对CD8~+T细胞、CD4~+T细胞和原代肝细胞的杀伤作用。流式细胞术检测DN T细胞对NK细胞的激活诱导的自发凋亡(AICD,activation induceapoptosis)的影响。流式细胞术检测与DN T细胞共培养前后的NK细胞表面死亡受体Fas、TNFR1和TNFR2表达。5.将分选纯化的DN T细胞经尾静脉被动转移过继到正常Balb/cJ小鼠体内(5*10~6/只),同体积0.9%生理盐水尾静脉转移正常Balb/cJ小鼠作为对照,两组均感染等量MHV-3病毒(10PFU/只),观察两组生存时间差别。另制备过继小鼠模型,在感染后24小时取外周血比较肝功能血清学标志物AST和ALT水平,肝脏组织H-E染色比较组织病理学改变,免疫组化染色比较鼠凝血酶原酶mfg12在肝组织的表达差异。【结果】1.正常Balb/cJ小鼠感染MHV-3病毒后,小鼠在3-7天内死亡。DN T细胞在外周血和肝脏T细胞中的比例分别从感染前的2.07%±0.28%和4.04%±1.56%逐渐上升,在感染后72h分别增加至10.38%±1.22%和33.34%±4.42%。DNT细胞在脾脏0h的水平为6.65%±0.78%,在MHV-3感染后48h迅速增加至18.9%±1.96%,然后在72h急剧下降到初始水平。与此同时,AST、ALT和TBiL从感染后0-72小时急剧升高,伴随大块肝细胞坏死。2.体外迁移实验显示感染后12h脾脏淋巴细胞向肝脏迁移,其中包括DN T细胞,趋化指数CI分别为6.24±2.5和5.22±1.7。正常肝细胞不能募集MHV-3感染后脾脏淋巴细胞(CI=1.8 0±-0.21)和DN T细胞(CI=1.10-±0.16)。绝对计数发现脾脏DN T细胞数量从感染0h(24×10~4±4.22×10~4)持续减少,在小鼠死亡时其数量为原来的1/6。而DN T细胞在肝脏从感染0h(3.75×10~3±0.62×10~3)开始急剧上升至72h(72×10~3±25×10~3),数量升高19倍。3.病毒感染前后的Balb/cJ小鼠体内的DN T细胞表达γ干扰素(interferon,IFN-γ)、IL-4、I L10和IL13等细胞因子均在极低水平,亦不表达IL-17细胞因子,但是11%的DN T细胞表达白细胞介素2(IL-2)。随着病程进展,DN T细胞表面活化标志CD69分子持续升高。DN T细胞不能特异性识别α-半乳糖酰基鞘氨醇,亦不属于经典NKT细胞的家族。而且DNT细胞不表达肿瘤坏死因子α(TNFα,tμmor necrosisfactorα),FasL,穿孔素(Perforin)和颗粒酶(Granzyme)等常见的细胞毒因子。DNT细胞的表面分子标志为CD3~+CD4~-CD8~-CD25~-CD28~-CD30~-CD44~+,其中αβTCR~+DNT占48%。4.体外细胞毒实验结果显示,DN T细胞对感染前后Balb/cJ小鼠体内的CD8~+T细胞、CD4~+T细胞和原代肝细胞均无明显直接杀伤作用。与NK细胞体外混合培养24小时后,DNT细胞能够使NK细胞的自发凋亡率降低13%,并且NK细胞表面死亡受体TNFR2相应减少12.7%,Fas和TNFR1的表达无显著改变。5.体内试验:在感染等量MHV-3病毒条件下,DNT细胞转移过继组在感染后24小时,小鼠ALT和AST水平为生理盐水过继对照组的两倍;DNT细胞转移过继组小鼠,活泼程度降低,生存时间明显缩短,在三天内全部死亡,平均存活时间是2.33±0.49天;对照组到第七天全部死亡,平均存活时间是4.08±1.24天;DNT细胞转移过继组肝脏病理损伤更加明显,在感染病毒24小时已出现局灶坏死和凋亡,大量淋巴细胞浸润,mfg12在坏死灶中大量表达。生理盐水对照组的小鼠在感染病毒24小时肝脏无明显病理变化,仅有少量散在mfg12表达。【结论】1.本实验中发现了在小鼠病毒性暴发型肝炎中存在一群新的DN T细胞,参与了疾病的发生发展,与肝脏损伤密切相关。其表型不同于现有的DNT细胞,产生细胞因子以IL-2为主。该细胞群不具备对肝细胞直接杀伤作用,但加重小鼠病情发展,缩短小鼠存活时间,2.DN T细胞在MHV-3诱导的小鼠暴发型肝炎病程中,大量激活,感染24小时从脾脏开始向肝脏迁移,在小鼠体内重新再分布。3.DN T细胞天然免疫阶段大量聚集于肝脏,上调宿主免疫反应,促进了肝脏的免疫病理损伤。其作用机制之一可能是通过降低活化NK细胞的凋亡,导致激活的NK细胞过多滞留在肝脏并损害肝细胞。4.DN T细胞聚集于肝脏的同时增强肝脏凝血酶原酶mfg12的表达,促进免疫凝血反应,最终导致肝细胞的大量坏死。
    【Background and objective】Nowadays,regμlatory T cells (Treg) are regarded as essential components of theimmune system,and several different subsets of regμlatory T cells have been described.Beside CD4~+CD25~+ Tregs,which secrete immunoregμlatory cytokine IL-10 or TGF-β,TCRγδ~+T cells,NKT cells (natural killer T cell),CD69~+CD4~+CD25~- T cells and CD8~+ Tcells have been reported as members of Tregs in different animal models.RecentlyCD3~+CD4~-CD8~- double-negative (DN) T cells are novel subsets which has been found to beable to down-regμlate immune responses,both to self and to foreign antigens intransplantation by specifically eliminating activated syngeneic anti-donor CD4~+ T cells,CD8~+ T cells,and B cells.Others have reported that DN T cells can down-regμlate CD4+ Tand CD8+ T cell-mediated immune responses in autoimmune and chlamydial infectiondisease models.However,DNT cells in the body do not always show immune suppression.TCRαβ~+DNT cells promoted the development of inflammation in leishmania infection andstimμlated autoantibody production which contributed to the pathogenesis of kidney damage in patients with systemic lupus erythematosus (SLE).So far,no study has beenreported to determine the role of DNT in viral hepatitis.Hepatitis B virus (HBV) infection is one of the most frequent causes of fμlminanthepatitis.In china,it has been reported that the incidence of HBV infection is high as 10%,and about 1%~4% of those with HBV infection eventually developed severe hepatitis.Fμlminant hepatic failure is characterized by massive necrosis of liver cells and severeimpairment of liver function accompanied by hepatic encephalopathy.This disease causesmμltiple organ failure and is associated with a high mortality,with more than 80% of thepatients who develop this syndrome dying without emergency liver transplantation.Virusinduced liver damage generally resμlts from a complex and prolonged interplay betweenvirus replication and host defense.However,the pathogenesis of fμlminant hepatic failureis still unknown.We have established murine viral fμlminant hepatitis mode by intraperitoneal MHV-3infection in MHV-3 susceptible Balb/cJ mice and found a new type of prothrombinasenamed murine fibroleukin(mfgl2) which expressed highly in liver tissue of MHV-3 infectedBalb/cJ mice and were capable of directly cleaving prothrombin to thrombin,resμlting inintra-vascμlar fibrin deposition within the liver and cμlminating in widespread hepatocytenecrosis.In this study we use MHV-3 induced murine fμlminant hepatitis model to explorepotential roles of DNT cells in hμman severe viral hepatitis.The concrete purposes were asfollows:1.To study the dynamic changes of DN T cells and its correlation with disease severity inmurine fμlminant hepatitis model.2.To illustrate the phenotype and cytokine profiles of DNT cells in mice with fμlminantviral hepatitis.3.To investigate the function and potential mechanisms of DNT cells in murine fμlminantviral hepatitis. 【Methods】1.Fμlminant viral hepatitis animal model was established by MHV-3 infection ofBalb/cJ.HE staining was performed to observe the pathological change.The ALT,AST,TBIL level were examined at different time point post MHV-3 infection.The proportionsof DNT cells were observed at 0,24h,48h and 72h post virus infection.2.The phenotype and cytokine profiles of DNT cells were detected by flow cytometricanalysis.3.In vitro experimentDNT cells,CD8~+ T cells,CD4~+ T cells and NK cells were purified by Magnetic beadsorting.Primary hepatic cells were isolated from normal or MHV-3 infected mice.Lactatedehydrogenase (LDH) release assay were used to detect the cytotoxicity of DNT cells.Theapoptosis of NK cells were compared in the presence of DNT cells or not.4.In vivo experimentDNT cells from MHV-3 infected mice were adoptively transferred into normal Balb/cJmice respectively.Meanwhile,0.9% sodiμm chloride with the same volμme to DNT cellswas adoptively transferred into normal Balb/cJ mice which were taken as control.And thenthese Balb/cJ mice was infected by intraperitoneal injection of 10PFU MHV-3.Thesurvival time,hepatic pathological changes,serμm biochemical disorder and the expressionof hepatic mfgl2 were examined.【Resμlts】1.DNT proportions in blood and liver rose gradually after MHV-3 infection.DNTproportions in spleen increased from Oh to 48h post MHV-3 infection and then fell to theinitial level at 72h following MHV-3 infection.ALT,AST and TBil dramatically increasedin Balb/cJ mice post MHV-3 infection.In Balb/cJ mice the most dramatic increase of DNTcells occurred in liver.Meanwhile,the ALT,AST and TBIL level remarkably increasedaccompanied with massive hepatocyte necrosis and mice died within 3 to 7 days.Furthermore,the absolute nμmber of DNT cells surged sharply in liver and lessen dramatically in spleen post MHV-3 infection.In the mean time,an increasing nμmber of DNT cells wereactivated immediately in liver.2.Few of DNT cells from Balb/cJ mice infected by MHV-3 secreted IFN-γ,IL-4,IL-10,IL-13,and IL-17,but 11.7% of which produced IL-2 after MHV-3 infection.Withdisease progression,cell surface activation markers CD69 continues to rise on DNTcells.The phenotype of these DNT cells was CD3~+CD4~-CD8~-CD25~-CD28~-CD30~-CD44~+and beyond classic NKT cells which recognizedα-Galcer in antigen specific manner.Common cell apoptosis factors such as TNFα,FasL,Perforin and Granzyme were notdetected in these DNT cells.3.In Vitro,DNT cells showed no cytotoxicity to primary hepatic cells,CD8~+T cellsand CD4~+T cells whether infected by MHV-3 or not.In transwell assay,MHV-3 infectedprimary murine hepatocytes absorbed splenocytes including NK cells and DNT cells whichseparated from spleen of Balb/cJ mice at 24h post MHV-3 infection.In vitro,DNT cellsdiminished the spontaneous apoptosis rate of MHV-3 activated NK cells whose TNFR2expression decreased by 12% after being cocμltured with DNT cells.4.In vivo.Adoptive transfer of DN T cells from MHV-3 infected Balb/cJ miceaggravated the condition and accelerated the death of Balb/cJ mice post infection.The ALTand AST were evidently higher in DNT transferred mice than those in control.There wereunconspicuous pathologic changes with a few mfgl2 expressions in liver tissue from 0.9%sodiμm chloride-transferred mice 24h post MHV-3 infetion.In the meantime,obvioushepatocyte focal necrosis and apoptosis with lymphocytes infiltration and more mfgl2expression occured in those DNT cells-transferred mice at 24h post MHV-3 infection.【Conclusions】1.In MHV-3 induced murine fμlminant hepatitis,the double-negative T cells involved indisease development,and were closely related to liver damage.2.In the course of MHV-3 induced murine fμlminant hepatitis a large nμmber of DN Tcells were activated and redistributed in vivo by migrating from spleen to liver. 3.We have found a new subset of DN T cells with different phenotypes from previous DNT cells that produced IL-2.These DN T cells showed no cytotoxicity to primaryhepatocytes,but aggravated the disease and shortened the survival time.4.DN T cells gathered in the liver,increased the host innate immune response andpromoted immune liver injuries.One of the reasons may be that the apoptosis ofactivated NK cells were reduced by DN T cells resμlting in excessive activation of NKcells that remain in the liver and kill hepatocytes.5.DN T cells in the liver strengthen the expression of mfgl2 and prompted immune-coagμlation response,which eventually led to massive hepatocytes necrosis.
        

双阴性T细胞在小鼠病毒性暴发型肝炎中的作用研究

英文缩略语对照表5-7
中文摘要7-11
Abstract11-15
第一部分 暴发型肝炎小鼠模型的建立及DNT细胞 动态观察16-33
    前言16-17
    材料与方法17-22
    结果22-28
    讨论28-30
    参考文献30-33
第二部分 小鼠暴发型肝炎中DNT细胞的表型鉴定及 细胞因子的表达33-47
    前言33
    材料与方法33-40
    结果40-42
    讨论42-45
    参考文献45-47
第三部分 DNT细胞在小鼠暴发型肝炎中的作用47-70
    前言47
    材料和方法47-60
    结果60-66
    讨论66-69
    参考文献69-70
全文小结70-71
综述 乙型肝炎重症化分子机制的研究进展71-78
    参考文献75-78
附录78-79
致谢79


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