【作者】 顾静；

【导师】 甘建和；

【作者基本信息】 苏州大学 ， 传染病学， 2013， 硕士

【摘要】 背景在我国，慢性乙型病毒性肝炎（CHB）是引起慢加亚急性肝衰竭（SACLF）的主要病因，有报道称CHB占SACLF所有病因的80%。目前认为，HBV感染患者病情进展和转归受诸多因素影响，其中宿主的免疫功能发挥了重要作用，细胞因子在调节免疫应答中扮演了关键角色。辅助性T细胞（Th细胞）根据分泌的细胞因子不同分为：Th0、Th1、Th2、Th3，目前研究最多的是Th1和Th2两个亚型。研究认为在病毒性肝炎感染中，Th1细胞有利于病毒的清除，而Th2细胞主要与组织损伤及慢性化有关。最近，CD4+Th细胞家族新型成员-Th17细胞亚群的出现改写了经典的Th1/Th2细胞反应模式。该细胞以高分泌IL-17为特征，具有很强的促炎症作用。2005年Kim等人发现了IL-32，该因子可由T细胞、NK细胞、单核细胞、上皮细胞产生，进一步研究表明其有促炎症因子的特性。IL-10是机体一种强有力的免疫和炎症抑制因子，由Th2细胞、巨噬细胞、树突状细胞（Dendritic cells，DC）和B细胞等产生，它可以减低T细胞活性，从而降低免疫反应的效应，导致机体清除病毒能力减弱。上述细胞及细胞因子在CHB中的变化已有报道，但其是否参与了HBV相关慢加亚急性肝衰竭（HBV-SACLF）的发病过程以及在肝衰竭病程中的动态变化趋势、与ALT的相关性研究未见报道。目的探讨HBV-SACLF患者外周血Th1、Th2、Th17及血清中IL-32、IL-10的动态变化趋势及其临床意义。方法采用流式细胞技术检测38例HBV-SACLF患者、46例肝衰竭前期患者、20例CHB患者、20例健康体检者（对照组），外周全血标本中Th1、Th2、Th17的表达水平，比较四组之间上述指标的差异；动态观察HBV-SACLF患者早、中、晚期Th1、Th2、Th17的变化趋势；比较感染组与非感染组、存活组与非存活组Th1、Th2、Th17表达水平的变化；分析Th1、Th2、Th17与ALT的相关性。同时采用ELISA检测上述四组血清中IL-32、IL-10的表达水平，并观察IL-32、IL-10在HBV-SACLF患者早、中、晚期的动态变化。结果1.HBV-SACLF组、肝衰竭前期组、CHB组、对照组Th1、Th2、Th17的表达水平分别为：①T h1：（22.58±3.86）%、（20.80±3.32）%、（5.23±1.51）%、（6.45±1.27）%；Th1比例在HBV-SACLF组与CHB组及对照组比较，均明显升高，差异具有显著性（P₂=0.04，P3=0.032）。②Th2：（1.73±0.64）%、（1.03±0.34）%、（6.15±0.97）%、（1.98±0.46）%；Th2比例在HBV-SACLF组与CHB组及对照组比较，均明显下降，差异具有显著性（P₂=0.007，P3=0.041）。③T h17：（3.48±0.80）%、（3.61±1.20）%、（1.09±0.4）%、（0.72±0.11）%；Th17比例在HBV-SACLF组与CHB组及对照组比较，均明显升高，差异具有显著性（P₂=0.003，P3<0.001）。2.HBV-SACLF早期、中期、晚期Th1、Th2、Th17的表达水平分别为：①Th1：（26.11±5.19）%、（22.45±4.06）%、（18.33±3.21）%；Th1比例在HBV-SACLF早期与中期，中期与晚期相比逐渐降低，两组之间比较差异均具有显著性（P1=0.001，P₂=0.004）。②T h2：（0.95±0.20）%、（1.66±0.41）%、（2.54±1.03）%；Th2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=0.048）。③Th17：（2.71±0.34）%、（3.39±0.73）%、（4.45±1.22）%；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=0.002，P₂=0.007）。3.HBV-SACLF感染组与非感染组Th1、Th2、Th17的表达水平分别为：①Th1：（23.59±3.91）%、（21.44±3.51）%；两组比较差异无显著意义。②T h2：（1.92±0.70）%、（1.76±0.55）%；两组比较差异无显著意义。③T h17：（4.65±1.33）%、（3.92±1.27）%，两组比较差异具有显著性（P=0.032）。4.HBV-SACLF存活组与非存活组Th1、Th2、Th17的表达水平分别为：①Th1：（20.78±3.37）%、（25.61±4.05）%；两组比较差异无显著意义。②T h2：（1.65±0.59）%、（1.88±0.63）%；两组比较差异无显著意义。③T h17：（3.21±1.02）%、（4.53±1.29）%，两组比较差异具有显著性（P=0.029）。5.对HBV-SACLF患者Th17、Th1、Th2与ALT进行Pearson相关性分析，结果发现Th17与ALT呈正相关（r=0.616，P=0.044），差异具有统计学意义；Th1、Th2与ALT无明显相关性。6. HBV-SACLF组、肝衰竭前期组、CHB组、对照组IL-32、IL-10的表达水平分别为：①IL-32：（500.98±152.33）pg/ml、（486.45±129.06）pg/ml、（281.72±99.28）pg/ml、（178.16±50.54）pg/ml；IL-32在HBV-SACLF组与CHB组及对照组比较，均显著升高，差异具有显著性（P₂=0.021，P3=0.033）。②I L-10：（2.82±1.03）pg/ml、（3.05±1.83）pg/ml、（13.15±3.37）pg/ml、（7.62±2.17）pg/ml；IL-10在HBV-SACLF组与CHB组及对照组比较，均显著下降，差异具有显著性（P₂=0.024，P3=0.019）。7. HBV-SACLF早期、中期、晚期IL-32、IL-10的表达水平分别为：①IL-32：（540.69±155.71）pg/ml、（498.43±135.56）pg/ml、（450.77±102.33）pg/ml；IL-32在HBV-SACLF早期与中期，中期与晚期相比逐渐降低，两组之间比较差异均具有显著性（P1=0.046，P₂=0.001）。②I L-10：（1.94±0.44）pg/ml、（2.83±0.97）pg/ml、（3.69±1.23）pg/ml；IL-10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=0.004，P₂=0.032）。结论Th1细胞和IL-32在HBV-SACLF患者中明显升高，随着病情发展呈逐渐下降趋势；Th2细胞和IL-10在HBV-SACLF患者中明显降低，其在HBV-SACLF早、中、晚期逐渐升高；Th17细胞在HBV-SACLF患者中明显升高，随着病情发展呈逐渐升高趋势；继发感染组Th17细胞较非感染组明显升高；存活组Th17细胞低于非存活组；且Th17细胞与肝细胞损害呈正相关。本文研究辅助性T细胞1、2、17及细胞因子IL-32、IL-10的紊乱是导致HBV-SACLF发生、发展的关键因素，通过检测其在肝衰竭早、中、晚期的动态变化可以判断机体的免疫状态，为临床的免疫调控治疗提供理论依据。更多还原

【Abstract】 Background In China, as a major cause of Subacute-on-chronic liver failure（SACLF）, CHB accounted for80%of all SACLF etiology. Currently it considered that theoutcome of HBV infection is influenced by many factors, and the host’s immune played asignificant role, and the cytokines also played a key role in regulating the immune response.According to the secretion of cytokines, T helper cells were divided into: Th0, Th1, Th2,Th3, Studies suggested that in viral hepatitis infection, Th1cells favored the removal of thevirus while Th2cells were primarily associated with tissue damage and chronic. Recently,a new effecter Th17belonging to CD4+subsets rewritted the classic Th1/Th2cell reactionmode, which was a strong pro-inflammatory factor characterized by secretion ofinterleukin17. In2005,Kim et al found IL-32,which produced by T cells, NK cells,monocytes, epithelial cells.Further studies showed it could promote the characteristics ofinflammatory cytokines. IL-10as a strong immune and inflammatory inhibitor wasproduced by Th2cells, macrophages, dendritic cells （Dendritic cells, DC）, and B cells, etc.It could reduce the activity of T cells and then reduced the immune response, making thebody to get rid of the virus diminished capacity. Above cells and cytokines had beenreported t in CHB, whether the dynamic changes of the above cells and cytokines in thehepatitis B-related SACLF （HBV-SACLF）in different stages and the correlation of theirbetween ALT had not been reported.Objective To investigate the expression，dynamic change and clinical significance ofTh1、Th2、Th17in peripheral blood and IL-32、IL-10in serum of the patients with SACLF during different periods.Methods Detected the ratio of Th1、Th2、Th17from peripheral blood cells in38HBV-SACLF patients,46pre-liver failure patients,20CHB patients and20health adultsby fluorescence-activated cell sorter; compared the above indicators among the four groups;observed dynamic changes of Th1、Th2and Th17during the nonage, metaphase andadvanced stage of HBV-SACLF; studied the expression level changes of Th1, Th2, ofTh17in infection group and non-infected group, survival group and non-survival group;analyzed the correlation of Th1, Th2,Th17and ALT. Detected the expression ofIL-32,IL-10from the serum of38HBV-SACLF patients,46Pre-liver failure patients,20CHB patients and20health adults by ELISA; observed dynamic changes of IL-32,IL-10during the nonage, metaphase and advanced stage of HBV-SACLF.Results1. The ratios of Th1, Th2,Th17on peripheral blood cells in38HBV-SACLF patients,46Pre-liver failure patients,20CHB patients and20health adults were as follows:①Th1：（22.58±3.86）%、（20.80±3.32）%、（5.23±1.51）%、（6.45±1.27）%;the ratios of Th1in HBV-SACLF group were significantly higher than CHB group and control group（P₂=0.04，P3=0.032）.②T h2：（1.73±0.64）%、（1.03±0.34）%、（6.15±0.97）%、（1.98±0.46）%; the ratios of Th2in HBV-SACLF group were significantly lower than CHB group andcontrol group（P₂=0.007，P3=0.041）③Th17：（3.48±0.80）%、（3.61±1.20）%、（1.09±0.4）%、（0.72±0.11）%,;the ratios of Th17in HBV-SACLF group were significantly higherthan CHB group and control group（P₂=0.003，P3<0.001）.2. The ratios of Th1, Th2and Th17cells in the nonage, metaphase and advanced stageof38HBV-SACLF patients were as follows:①T h1：（26.11±5.19）%、（22.45±4.06）%、（18.33±3.21）%;The ratios of Th1decreased gradually, There were also significantdifferences among the three periods（P1=0.001, P₂=0.004）.②Th2：（0.95±0.20）%、（1.66±0.41）%、（2.54±1.03）%；The ratios of Th2increased gradually, There were alsosignificant differences among the three periods（P1=0.003, P₂=0.048）.③T h17：（2.71±0.34）%、（3.39±0.73）%、（4.45±1.22）%; The ratio of Th17increased gradually, There were also significant differences among the three periods（P1=0.002, P₂=0.007）.3.The ratios of Th1,Th2,Th17cells between the infection group and the non-infectedgroup in HBV-SACLF patients were as follows:①T h1：（23.59±3.91）%、（21.44±3.51）%; The difference was not statistically significant between the two groups.②Th2：（1.92±0.70）%、（1.76±0.55）%; The difference was not statistically significant betweenthe two groups.③Th17：（4.65±1.33）%、（3.92±1.27）%; There was significantly differencebetween the two groups （P=0.032）.4. The ratios of Th1,Th2,Th17cells between the survival group and the death group inHBV-SACLF patients were as follows:①Th1：（20.78±3.37）%、（25.61±4.05）%; Thedifference was not statistically significant between the two groups.②T h2：（1.65±0.59）%、（1.88±0.63）%; The difference was not statistically significant between the two groups.③T h17：（3.21±1.02）%、（4.53±1.29）%; There was significantly difference between thetwo groups （P=0.029）.5. Correlation analysis of Th1,Th2,Th17and ALT of HBV-SACLF patients: Th17hadsignificant positive correlation with ALT（r=0.616， P=0.044）. Th1and Th2hadnon-significant correlation with ALT.6. The expression of IL-32, IL-10in38HBV-SACLF patients,46Pre-liver failurepatients,20CHB patients and20health adults were as follows:①IL-32：（500.98±152.33）pg/ml、（486.45±129.06）pg/ml、（281.72±99.28）pg/ml、（178.16±50.54）pg/ml; The ratiosof IL-32in HBV-SACLF group were significantly higher than CHB group and controlgroup（P₂=0.021，P3=0.033）.②IL-10：（2.82±1.03）pg/ml、（3.05±1.83）pg/ml、（13.15±3.37）pg/ml、（7.62±2.17）pg/ml; The ratios of IL-10in HBV-SACLF group weresignificantly lower than CHB group and control group （P₂=0.024，P3=0.019）.7. The ratios of IL-32, IL-10in the nonage, metaphase and advanced stage of38HBV-SACLF patients were as follows:①I L-32：（540.69±155.71） pg/ml、（498.43±135.56）pg/ml、（450.77±102.33）pg/ml; The ratios of IL-32decreased gradually,There were also significant differences among the three periods（P1=0.046, P₂=0.001）.②I L-10：（1.94±0.44）pg/ml、（2.83±0.97）pg/ml、（3.69±1.23）pg/ml; The ratios of IL-10 increased gradually, There were also significant differences among the three periods（P1=0.004, P₂=0.032）.Conclusion The Th1and IL-32in HBV-SACLF patients were significantly increasedand there was a gradual downward trend with the progression of the disease. Th2,Th17andIL-10were significantly higher and upward gradually with the progression of SACLF. Theratios of Th17in infection group were significantly higher than non-infected group;however, it was lower in survival group than death group and had significant positivecorrelation with ALT. In our studies, the disorders of Th1,Th2,Th17and IL-32, IL-10werekey factors in the development of HBV-SACLF. By detecting their dynamic changes inHBV-SACLF, we can judge patients’ immune status and offer theoretical basis for theclinical treatment of immune regulation.更多还原