【作者】 杨柳；

【导师】 甘建和；

【作者基本信息】 苏州大学 ， 传染病学， 2013， 硕士

【摘要】 背景乙型肝炎病毒(hepatitis B virus,HBV)感染机体，引起一系列复杂的免疫反应，其中细胞免疫是决定HBV感染机体后转归的重要因素。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免疫抑制作用。CD8~+T细胞在清除HBV过程中起重要作用，其活化依赖于CD4~+T细胞。糖皮质激素主要通过抑制细胞毒性T淋巴细胞及单核/吞噬细胞发挥作用，从而阻止肝细胞进一步损伤，但其在乙肝相关性肝衰竭中的应用仍然存在争议，尤其是对T淋巴细胞亚群的影响则鲜有报。白介素12（IL-12）是联系细胞免疫与体液免疫的重要细胞因子之一，然而在肝衰竭前期治疗中糖皮质激素对其影响未见相关报道。目的探讨HBV相关慢加亚急性肝衰竭（HBV-associated subacute-on-chronic liverfailure,HBV-SACLF）患者外周血CD4~+T细胞、CD8~+T细胞、Treg细胞变化趋势，并探讨糖皮质激素对肝衰竭前期的治疗机制。方法采用流式细胞仪检测30例HBV相关的慢加亚急性肝衰竭（HBV-SACLF）患者、15例慢性乙型肝炎患者(CHB)、12例健康对照者患者外周全血标本中CD4~+T细胞、CD8~+T细胞、Treg细胞的百分比，比较各组之间的差异；同时将肝衰竭患者分为感染组与非感染组，观察以上指标变化。同时将76例肝衰竭前期患者分为糖皮质激素治疗组（GC组）38例与非糖皮质激素治疗组（NG组）38例，监测以上指标。同时采用ELISA技术检测肝衰竭前期患者血清中IL-12的表达水平。结果1.HBV-SACLF组、CHB组及正常对照组CD4~+T、CD8~+T、Treg百分比分别为：1)CD4~+T：(28.06±6.86)%、(36.03±9.53)%、(45.47±0.68)%，HBV-SACLF组与CHB组、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=0.02，p2<0.001，p3=0.02)；2)CD8~+T：(29.10±3.64)%、(25.71±5.75)%、(20.10±5.80)%，CD8+T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，均明显降低，差异均有统计学意义(p1=0.02，p2<0.001，p3=0.019)；3)Treg：(2.55±0.57)~+、(3.69±1.08)~+、(3.21±0.12)~+；Treg比例在HBV-SACLF组较CHB组及正常组均明显下降，差异有统计学意义（p1=0.01，p2<0.001），CHB组较正常组升高，差异不明显（p3=0.113）。2.HBV-SACLF中感染组CD4+T、CD8+T、Treg百分比分别为（25.33±7.79）~+、（30.90±2.94）~+、（2.31±0.38）~+，非感染组CD4+T、CD8+T、Treg百分比分别为（31.19±3.91）~+、（26.93±3.16）~+、（2.82±0.63）~+，两组相比较均有明显差异（p1=0.014，p2=0.001，p3=0.015）。3.GC组与NG组治疗前CD4+T、CD8+T、Treg百分比均无明显差异，GC组治疗后CD4+T、CD8+T、Treg百分比分别为（35.44±5.36）~+、（24.40±5.68）~+、（3.09±0.42）~+，较治疗前（28.72±3.79）~+、（28.75±4.72）~+、（2.69±0.69）~+差异均有统计学意义（p1<0.001，p2=0.001，p3=0.004）；NG组治疗后CD4+T、CD8+T、Treg百分比分别为（31.35±5.44）~+、（28.6±6.62）~+、（2.82±0.51）~+，较治疗前（28.27±3.51）~+、（29.44±5.29）~+、（2.72±0.70）~+相比，仅CD4+T差异有统计学意义（p1=0.004，p2=0.435，p3=0.482）。4.GC组与NG组治疗前IL-12无明显差异，GC组治疗后IL-12为（28.98±7.81）pg/ml，较治疗前（50.54±18.27）pg/ml相比差异均有统计学意义（p<0.001）；NG组治疗后IL-12为（46.33±22.22）pg/ml较治疗前（52.12±17.61）pg/ml相比，差异无统计学意义（p=0.212）。5.GC组中病情重症化率为13.16~+，NG组中病情重症化率为34.21~+，两组差异有统计学意义（P<0.05）。结论1.HBV-SACLF患者外周血CD4+T、Treg比例下降，CD8+T升高，表明肝衰竭患者存在严重的细胞免疫失衡。2.HBV-SACLF中感染组较非感染组相比CD4+T、Treg比例下降明显，CD8+T明显升高，说明感染加重了机体免疫失衡，不利于病情恢复。3.GC组较NG组相比，外周血CD4+T、Treg比例较治疗前明显上升，CD8+T较治疗前明显下降，而且GC组重症化率明显降低，表明GC通过调节机体免疫功能促进病情恢复。4.GC组治疗后IL-12水平较NG组明显下降，表明GC可以抑制IL-12的产生，从而减轻机体免疫反应。更多还原

【Abstract】 Background The infection of hepatitis B virus causes a series of complex immuneresponse,and the cell immunity is an important factor for the outcome. Regulatory T cells(Treg) belonging to CD4~+subsets have the function of immune regulatory includingimmune incompetence and immunosuppressive by contacting among cells and secretion ofinhibitory cytokines,which also have the function of anti-inflammatory. CD8~+T cells playan important role in the process of clearing HBV.The activation of CD8~+T cells depends onCD4~+T cells.Glucocorticoid can reduce hepatocyte injury through inhibiting the productionof monocyte/macrophage and B cells.But it is still controversial about the application ofglucocorticoid on severe hepatitis B.At the same time, the effect of glucocorticoid on T cellsubsets is rarely reported and the mechanism remains unknown. Interleukin12（IL-12) isone of the most important cytokines which associated with cellular immunity and humoralimmunity.It has not been reported that the effect of glucocorticoid treated pre-liver failure.Objective To explore the changes of CD4~+T,CD8~+T and Treg in peripheral blood of patientswith HBV-associated subacute-on-chronic liver failure（HBV-SACLF）,and the mechanismof glucocorticoid on pre-liver-failure.Methods Detected the ratio of CD4~+T,CD8~+T and Treg from peripheral blood cells byfluorescence-activated cell sorter in30HBV-SACLF patients,15CHB patients12healthadults.HBV-SACLF patients were divided into infection group and non-infectiongroup,and detected above indicators;76pre-liver-failure patients were divided intoglucocorticoid group (GC group) and non-glucocorticoid (NG group),and detected aboveindicators.Detected the expression of IL-12from the serum by ELISA. Results1.The ratios of CD4~+T,CD8~+T and Treg on peripheral blood cells in30HBV-SACLFpatients,15CHB patients and12health adults were as follows:1)CD4~+T:(28.06±6.86)%,(36.03±9.53)%,(45.47±0.68)%.The ratios of CD4~+T in CHB group weresignificantly higher than HBV-SACLF group, and lower than control-group(p1=0.02,p2<0.001,p3=0.02);2)CD8~+T:(29.10±3.64)%,(25.71±5.75)%,(20.10±5.80)%.The ratios ofCD8~+T in CHB group were significantly lower than HBV-SACLF group,whether higherthan control group(p1=0.02,p2<0.001,p3=0.019);3)Treg:(2.55±0.57)%,(3.69±1.08)%,(3.21±0.12)%.The ratio of Treg in HBV-SACLF group were significantly lower comparedwith CHB group and normal group(p1=0.01,p2<0.001). There were no significantdifferences between CHB group and normal group(p3=0.113).2.The ratios of CD4~+T,CD8~+T and Treg in infection group of HBV-SACLF patients wereas follows:(25.33±7.79)%,(30.90±2.94)%,(2.31±0.38)%;and in non-infection group ofHBV-SACLF patients were as follows:(1.19±3.91)%,(26.93±3.16)%,(2.82±0.63)%.Therewere significant differences between the two groups(p1=0.014,p2=0.001,p3=0.015).3.There were no significant differences between the GC group and NG group beforetreatment.The ratios of CD4~+T,CD8~+T and Treg in GC group after treatment were asfollows:(35.44±5.36)%,(24.40±5.68)%,(3.09±0.42)%.There were significant differencescompared with pre-therapy(p1<0.001,p2=0.001,p3=0.004).The ratio of CD4~+T in NGgroup after treatment was (31.35±5.44)%.There were significant differences comparedwith pre-therapy(p1=0.004).And the changes on CD8~+T and Treg were notclearly(p2=0.435,p3=0.482).4.There were no significant differences of IL-12between the GC group and NG groupbefore treatment.The value of IL-12in GC group was(28.98±7.81)pg/ml aftertreatment.There was significant difference compared with pre-therapy(p<0.001).The valueof IL-12in NG group was(46.33±22.22)pg/ml after treatment.There was no significantdifference compared with pre-therapy(p=0.212).5.The incidence rate of severe hepatitis in GC group was13.16%and that in NG group was 34.21%, there was significant difference between two groups (p<0.05).Conclusion1.The ratios of CD4~+T and Treg on peripheral blood cells in HBV-SACLF patients weredramatic decline.The ratio of CD8~+T was markedly increased.It indicated that the hepaticfailure patients had severe immune imbalance.2.The ratios of CD4~+T and Treg in infection group of HBV-SACLF patients were lowerthan non-infection group.The ratio of CD8~+T was higher than non-infection group.Itindicated that the infection increased immune imbalance,and make disease to worse.3.The ratios of CD4~+T and Treg in GC group were higher than NG group,and The ratio ofCD8~+T was lower than NG group.And the incidence rate of severe hepatitis in GC groupwas significant decreased which indicated that glucocorticoid promoted the recovery ofillness through Immunomodulatory.4.The expression of IL-12in GC group was lower than NG group after treatment.Itindicated that glucocorticoid relieved inflammation through inhibiting the production of IL-12.更多还原