【作者】 刘晓燕；

【导师】 王慧芬；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2009， 博士

【摘要】 目的:乙型肝炎病毒感染慢加急性肝衰竭(HBV-associated acute-on-chronic liver failure,HBV-ACLF)病情危重、发展迅速、病死率高达70～80%,是传染病死亡的重要疾病之一。其发病机制不是十分明确。目前研究多集中于“免疫正性调控”的研究,对于负性调节分子在HBV-ACLF中的表达情况未见相关报道。本文研究了HBV-ACLF过程中患者体内T细胞亚群上免疫共刺激负性调控分子——程序性死亡分子(Programmed death-1,PD-1)及其配体PD-L1的表达变化以及与病情预后之间的关系,试图阐明HBV-ACLF过程中PD-1/PD-L1表达对病情的变化影响。方法:选择60例HBV-ACLF患者、15例肝炎肝硬化乙型活动性患者及15例健康人为研究对象(实验选择的病例ALT或AST均大于正常值的两倍)。临床数据的收集:实时定量PCR检测患者的HBVDNA,收集临床检验数据:血常规、肝功、凝血酶原活动度等指标;根据入组时病情分为早、中、晚三期,根据疾病不同转归分为好转、无效、死亡。应用流式细胞仪检测外周血CD8~+T细胞上相关分子:1)T淋巴细胞上的PD-1、CD28、CD95、CD45RA、CCR7;2)CD14~+细胞上的PD-L1;3)CD8~+细胞上的穿孔素、颗粒酶A、颗粒酶B、CD107a。应用CBA(CytometricBead Array)试剂盒动态检测外周血细胞因子,包括白介素-2(interleukin-2,IL-2)、IL-4、IL-6、IL-10,肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)等。取10例HBV-ACLF患者的肝脏行免疫组化染色分析,另各取10例肝炎肝硬化患者、6例健康人作为对照组。了解ACLF、肝炎肝硬化患者肝脏上CD4~+T细胞、CD8~+T细胞、PD-1及其配体PD-L1的表达情况。结果:HBV-ACLF患者CD8~+T细胞上PD-1表达上调(17.31%±11.28%),较肝硬化患者(11.18%±5.38%)和健康人(5.96%±4.19%)显著增高;根据Meld评分分组,Meld≥20分组患者PD-1阳性表达率(20.98%±12.28%)比＜20分组患者(13.61%±8.23%)明显增高,组间具有显著性差异(P＜0.05);好转组PD-1表达率(12.89%±7.18%)低于无效组(21.19%±11.21%),组间具有显著性差异(P＜0.05);早期(13.27%±7.2%)、中期(18.86%±10.31%)、晚晚(29.51%±19.59%)三期PD-1表达率呈上升趋势,组间均有显著性差异(P＜0.05)。CD14~+T细胞上PD-L1在疾病早期表达低,随着病情的加重表达成上升趋势,早期(4.19%±3.51%)分别与中期(9.24%±6.92%)、晚期(7.05%±5.34%)有显著性差异(P＜0.05);好转组与死亡组有显著性差异(P＜0.05)。CD8~+T细胞上CD95在死亡组表达高于好转组与无效组,但无显著性差异。代表CD8~+T细胞杀伤能力的穿孔素、颗粒酶及CD107a在死亡组表达升高,与其它组间有显著性差异(P＜0.05),但穿孔素、颗粒酶及CD107a阳性细胞上PD-1阳性表达率非常低。随着病情的加重致炎因子IFN-γ、TNF-α表达增多,可能促进了PD-1的上调表达。抑炎因子IL-10无明显变化。此外,HBV-ACLF和肝硬化者在肝组织汇管区及炎症区PD-1表达增强,肝细胞上PD-L1表达上调。结论:PD-1在HBV-ACLF中外周血CD8~+T细胞上表达上调,且与病情严重程度成正比。HBV-ACLF汇管区及炎症区PD-1及肝细胞上PD-L1表达上调;PD-1/PD-L1的升高可能与免疫功能紊乱、病情加重有关。HBV-ACLF过程中效应性CD8~+T细胞上PD-1表达率较其它CD8~+T细胞低,PD-1对该类细胞的功能抑制及促凋亡作用减弱,可能与病情的加重有关。更多还原

【Abstract】 Characteristics of PD-1/PD-L1 expression in HBV-associated acute-on-chronic liver failureObjectiveTo investigate the expression pattern of programmed death-1(PD-1) and its ligand (PD-L1) in HBV-associated acute-on-chronic liver failure(HBV-ACLF) patients and the relationship between PD-1 expression and the prognosis of HBV-ACLF.To elucidate the impacts of PD-1/PD-L1 on the progression of HBV-ACLF. Methods60 HBV-ACLF,15 liver cirrhosis(LC) and 15 healthy control(HC) were enrolled in this study.For HBV-ACLF,subjects were followed up for one month at least and venous blood was colleted every week.Clinical parameters were collected accordingly, including HBV viral load,ALT,AST,TBIL,PA,WBC et al.We analyzed the expressions of PD-1,CD95,CD45RA,CCR7,Perforin,granzyme A,granzyme B, CD107a on CD8~+T lymphocytes and the expression of PD-L1 on monocytes in peripheral blood by using flow cytometry.CBA(Cytometric Bead Array) kit was employed to measure the concentrations of IL-2(interleukin-2),IL-4,IL-6,IL-10,TNF-αand IFN-γin serum.Paraffin-embedded liver sections from HBV-ACLF liver tissue(10 cases),biopsies of LC patients(10 cased),and healthy donor liver sections(6 cases) were used for immunohistochemical staining,including CD4,CD8,PD-1 and PD-L1.ResultsThe PD-1 expression in HBV-ACLF patients(17.31%±11.28%) was significantly elevated compared with those in HC(11.18%±5.38%) and LC subjects(5.96%±4.19%). PD-1 also up-regulated in the group whose meld score≥20(20.98%±12.28%),compared with＜20 group(13.61%±8.23%).(P＜0.05).PD-1 expression was lower in improved group(12.89%±7.18%) than that in non-improved group(21.19%±11.21%).(P＜0.05), and increased from early stage(13.27%±7.2%),middle stage(18.86%±10.31%) to late stage(29.51%±19.59%).(P＜0.05).Moreover,PD-L1 expression on monocytes was positively correlated with disease progression.(P＜0.05) Both PD-1 and CD95 expressions were higher in dead group than those in improved and non-improved groups.Perforin,granzymes and CD107a expressions on CD8~+T cells significantly increased in dead group compared with those in improved and non-improved groups(P＜0.05).However,PD-1 expressions on these cells were lower,compared with normal persons.ConclusionsThe expression of PD-1 in HBV-ACLF patients was positively correlated with disase progression.PD-1 expression was increased in portal area and PD-L1 was increased on hepatocytes in liver section of HBV-ACLF patients.For HBV-ACLF patients,the PD -1 expression on effector CD8~+ T cells was lower than those in other cells,accounting for the failure in controlling immune injury in liver.更多还原