【作者】 胡凤霞；

【导师】 甘建和；

【作者基本信息】 苏州大学 ， 传染病学， 2012， 硕士

【摘要】 背景我国慢加亚急性肝衰竭病死率高达60％-80％。其发病机制复杂，目前认为宿主免疫调节的紊乱是造成肝脏病变的重要原因。其中T细胞亚群介导的细胞免疫以及细胞因子网络的调控异常起着重要的作用。最近，一种新的效应CD4+T细胞亚群Th17被广泛关注，具有很强的促炎症作用，以分泌白介素17(IL-17)为特征。调节性T细胞(Treg)是体内重要的具有免疫调节功能的CD4+T细胞亚群，具有免疫无能和免疫抑制两大功能特征，可通过细胞间的直接接触、分泌抑制性细胞因子等多种方式发挥抑制作用，具有抗炎症作用。细胞毒性T细胞(CD8+CD28+即CTL)在抗病毒、自身免疫等方面起重要作用，主要是通过细胞毒机制、非细胞毒机制两种途径特异性地杀伤靶细胞。Th17、Treg及CTL在慢性乙型病毒性肝炎中的研究已见报道，但上述三者是否参与了乙肝相关慢加亚急性肝衰竭(HBV-SACLF)发病的过程及在肝衰竭不同阶段的动态变化、三者之间的相关性研究尚未见报道。目的探讨HBV-SACLF患者外周血Th17、Treg、CTL细胞的动态变化趋势、三者之间的相关性及与预后的关系。方法采用流式细胞仪检测39例HBV-SACLF患者、20例慢性乙型肝炎患者(CHB)、15例正常人，外周全血标本中Th17、Treg及CTL的比例，比较三组之间上述指标的差异；动态观察HBV-SACLF患者早期、中期、晚期Th17、Treg、CTL的变化趋势；分析Th17、Treg、CTL细胞亚群间是否具有相关性；比较存活组与非存活组Th17、Treg、CTL比例与预后的关系。同时采用ELISA技术检测上述三组血清中IL-17的表达水平，并分析HBV-SACLF患者早期、中期、晚期IL-17的动态变化。结果1.HBV-SACLF组、CHB组及正常对照组Th17、Treg、CTL比例分别为：1)Th17：(4.04±1.22)%、(1.94±0.43)%、(1.01±0.25)%；Th17比例在HBV-SACLF组与CHB组及正常对照组比较，CHB组与正常组比较，均明显升高，差异均具有显著性(p1=0.03，p2<0.001，p3=0.034)。2)Treg：(3.79±1.49)%、(8.25±1.23)%、(5.76±1.15)%；Treg比例在HBV-SACLF组较CHB组及正常组均明显下降，CHB组较正常组明显升高，差异均具有显著性(p1<0.001，p2=0.006，p3=0.007)。3)CTL：(21.12±5.35)%、(13.61±1.41)%、(16.73±1.20)%；CTL比例在HBV-SACLF组较CHB组及正常组均明显上升，CHB组较正常组明显下降，两组之间比较差异均具有显著性(p1<0.001，p2=0.012，p3=0.009)。2.HBV-SACLF早期、中期、晚期Th17、Treg、CTL的比例分别为：1)Th17：(3.05±0.71)%、(4.07±0.90)%、(5.18±1.10)%；Th17比例在HBV-SACLF早期与中期，中期与晚期相比逐渐升高，两组之间比较差异均具有显著性(p1=0.003，p2=0.009)。2)Treg：(5.18±0.68)%、(3.99±0.72)%、(1.87±0.64)%；Treg比例在HBV-SACLF早期与中期，中期与晚期比较逐渐下降，差异均具有显著性(p1=0.001，p2=0.000)。3)CTL：(22.62±4.99)%、(19.08±3.63)%、(14.48±4.39)%；CTL比例在HBV-SACLF早期与中期，中期与晚期比较逐渐下降，差异均具有显著性(p1=0.046，p2=0.003)。3.HBV-SACLF存活组与非存活组Th17比例分别为：(3.44±0.97)%、(4.56±1.20)%；两组之间比较差异具有显著性(P=0.003)。且Th17与预后具有明显正相关性(r=0.501，P=0.029)。4.对HBV-SACLF患者中Th17、Treg及CTL进行相关性分析发现：Th17与Treg存在明显的负相关性(r=-0.532，P<0.001)；Treg与CTL存在显著的正相关性(r=0.339，P=0.035)；Th17与CTL无相关关系(r=-0.246，P=0.131)。5.HBV-SACLF组、CHB组及正常对照组IL-17细胞因子的表达水平分别为：289.2±83.3、153.3±79.6、62.5±28.5(pg/ml)，HBV-SACLF组与CHB组及正常对照组之间差异均有显著性意义(p1=0.019，p2=0.000)。6.HBV-SACLF早期、中期、晚期IL-17细胞因子的表达水平分别为：214.7±47.5、267.7±56.8、317.4±88.1(pg/ml)，在早期与中期、中期与晚期之间差异均有显著性(p1=0.011，p2=0.007)。结论HBV-SACLF患者外周血Th17细胞比例和IL-17水平明显升高，且在肝衰竭的进展过程中持续升高，与预后具有明显的正相关性；Treg细胞比例在HBV-SACLF时下降，且在肝衰竭进展过程中持续下降，与Th17之间具有明显的负相关性；CTL细胞比例在HBV-SACLF时上升，在肝衰竭进展过程中持续下降，与Treg之间具有明显的正相关性。表明机体内Thl7、Treg及CTL细胞比例紊乱可能是导致HBV-SACLF发生和发展的关键因素，其在外周血中的动态变化可以判断机体的免疫状态，对于指导临床治疗和判断预后具有一定的价值。更多还原

【Abstract】 Background The fatality rate of Subacute-on-chronic liver failure (SACLF) wasabout60-80%. Its pathogenesis was complex, and it was believed that immunoregulatorydisorder leading to liver lesions was important. The abnormal immune of lymphocytes andregulation of cytokine network mediated by subpopulation of T lymphocytes played animportant role in the process. Recently, a new effector Th17belonging to CD4+subsetswas concerned widely, which was a strong pro-inflammatory factor characterized bysecretion of interleukin17(IL-17). Regulatory T cells (Treg) belonging to CD4+subsetshad the function of immune regulatory including immune incompetence andimmunosuppressive functions by contacting among cells and secretion of inhibitorycytokines,which also had the function of anti-inflammatory. Cytotoxic T lymphocytes(CD8+CD28+, CTL) played an important role in anti-virus and autoimmune, mainlythrough cytotoxic mechanism and non-cytotoxic mechanism to kill target cells specifically.The study about Th17, Treg and CTL in CHB had been reported, whether the dynamicchanges of the above three factors involved in the hepatitis B-related SACLF(HBV-SACLF) in different stages the and the correlation of each other had not beenreported.Objective To explore the dynamic changes and correlation of Th17, Treg and CTL inperipheral blood of patients with HBV-SACLF and study the relationship with prognosis.Methods Detected the ratio of Th17, Treg and CTL from peripheral blood cells in39HBV-ACLF patients,20CHB patients and15health adults by fluorescence-activated cell sorter; compared the above indicators among the three groups; observed dynamicchanges of Th17, Treg and CTL during the nonage, metaphase and advanced stage ofHBV-SACLF; studied the correlation of prognosis of Th17, Treg and CTL cells betweensurvival group and death group. Detected the expression of IL-17from the serum of39HBV-SACLF patients,20CHB patients and15health adults by ELISA; observed dynamicchanges of IL-17during the nonage, metaphase and advanced stage of HBV-SACLF.Results1. The ratios of Th17, Treg and CTL on peripheral blood cells in39HBV-ACLFpatients,20CHB patients and15health adults were as follows:1)Th17:(4.04±1.22)%,(1.94±0.43)%,(1.01±0.25)%; The ratios of Th17in CHB group were significantlyhigher than control group, whether lower than HBV-SACLF group (p1=0.03, p2<0.001,p3=0.034).2)Treg:(3.79±1.49)%,(8.25±1.23)%,(5.76±1.15)%; The ratio of Treg inHBV-SACLF group were significantly lower compared with CHB group and normal group(p1<0.001, p2=0.006, p3=0.007).3)CTL:(21.12±5.35)%,(13.61±1.41)%,(16.73±1.20)%; The ratio of CTL in HBV-SACLF group were significantly higher compared withCHB group and normal group(p1<0.001, p2=0.012, p3=0.009).2. The ratios of Th17, Treg and CTL cells in the nonage, metaphase and advancedstage of39HBV-SACLF patients were as follows:1)Th17:(3.05±0.71)%,(4.07±0.90)%,(5.18±1.10)%; The ratios of Th17increased gradually, There were also significantdifferences among the three periods(p1=0.003, p2=0.009).2)Treg:(5.18±0.68)%,(3.99±0.72)%,(1.87±0.64)%; The ratios of Treg decreased gradually, There were alsosignificant differences among the three periods(p1=0.001, p2=0.000).3)CTL:(22.62±4.99)%,(19.08±3.63)%,(14.48±4.39)%; The ratio of Th17increased gradually, Therewere also significant differences among the three periods(p1=0.046, p2=0.003).3. The ratios of Th17cells between the survival group and the death group inHBV-ACLF patients were as follows:(3.44±0.97)%,(4.56±1.20)%; which had astatistical significance(P=0.003). Th17had positive correlation with prognosis(r=0.501,P=0.029). 4. Correlation analysis of Th17, Treg and CTL of HBV-SACLF patients: Th17hadsignificant negative correlation with Treg (r=-0.532, P <0.001); Treg had significantpositive correlation with CTL (r=0.339, P=0.035); Th17had non-significant correlationwith CTL(r=-0.246, P=0.131).5. The expression of IL-17in39HBV-ACLF patient,20CHB patients and15healthadults were as follows:289.2±83.3,153.3±79.6,62.5±28.5(pg/ml); There were alsosignificant differences among the three periods(p1=0.019, p2=0.000).6.The ratios of IL-17in the nonage, metaphase and advanced stage of39HBV-SACLF patients were as follows:214.7±47.5,267.7±56.8,317.4±88.1(pg/ml);There were also significant differences among the three periods(p1=0.011, p2=0.007).Conclusion The expression of Th17and IL-17obviously steped up in HBV-SACLFpatients, which escalated in the progression of liver failure, had a significant positivecorrelation with prognosis; Treg decreased in HBV-SACLF and declined gradually in theprocess of liver failure, Treg had a significant negative correlation with Th17; CTLincreased in the proportion of HBV-SACLF and declined gradually in the process of liverfailure, CTL had a significant positive correlation with Treg. Dynamic changes of Th17,Treg and CTL from peripheral blood may be in favour of evaluating immune state, whichwas also profit for the guidance of treatment and prognosis evaluation in HBV-SACLFpatients.更多还原