滤泡调节性T细胞对HBV相关慢加急性肝衰竭患者高表达抗可溶性肝抗原抗体的影响

发布时间：2018-03-01 17:20

  本文关键词： HBV相关慢加性急肝衰竭 滤泡调节性T细胞 肝抗原自身抗体 自身免疫　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景肝衰竭是临床常见的严重肝病症候群,病情严重、并发症多、治疗困难,在我国病死率高达60～80%。我国肝衰竭的病因主要为乙型肝炎病毒(hepatitis B virus,HBV)感染,目前临床表现主要以慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)为主。在我国急性肝衰竭(acute liver failure,AHF)和亚急性肝衰竭呈现减少趋势,而慢加急性肝衰竭和慢性肝衰竭呈增加趋势。HBV 相关慢加急性肝衰竭(HBV-related acute-on-chronic liver failure,H-A)的发病过程中,机体针对HBV的特异性免疫和非特异性免疫状态,与其他慢性HBV感染状态相比,发生了很大的变化。其中机体体液免疫方面也发生了很多的变化,如在H-A患者体内其表面抗原(Hepatitis B virus surface antigen,HBsAg)水平较慢性乙型肝炎(chronic hepatitis B,CHB)低,并且发生了 HBeAg/抗HBe的血清学转换,甚至在很多患者体内会出现自身抗体谱转阳的情况。研究中发现在慢性HBV感染过程中出现了大量自身抗体的转阳,包括抗核抗体、抗线粒体抗体、抗平滑肌抗体等。其中抗可溶性肝抗原抗体(anti-soluble liver antigen antibody,anti-SLA/抗SLA)是肝抗原自身抗体的类型之一,有研究表明抗SLA在CHB、代偿性肝硬化、非代偿性肝硬化、肝细胞癌(hepatocellular carcinoma,HCC)、急性肝衰竭(acute liver failure,AHF)中的阳性率分别为2.2%、3.5%、1.7%、2.6%、21.9%。但是,目前还没有关于抗SLA在H-A中的研究,而且为什么肝衰竭的患者中自身抗体表达上调,其机理也不清楚。抗体的产生是精细而复杂的过程。滤泡辅助性T细胞(Follicular T helper cells,Tfh细胞)辅助B细胞分化为浆细胞、产生抗体、及抗体类别转换。二者相互作用,在体液免疫应答中发挥重要作用。研究显示在自身免疫疾病中,Tfh细胞及其亚群频数发生变化,同时B细胞处于功能失调状态。在生发中心(Germinal Center,GC)的滤泡调节性 T 细胞(FollicularTregulatory cells,Tfr细胞),主要参与生发中心的反应,抑制Tfh细胞和B细胞,避免自身抗体的产生,与自身免疫性疾病相关。因此本文旨在研究Tfr细胞对H-A患者血浆抗SLA异常表达的影响。目的本研究是以HBV感染自然史的横向人群为研究对象,主要研究H-A患者表达自身抗体SLA的程度,以及高表达抗SLA与Tfr细胞频数和功能的关系。方法1.研究对象26例H-A为本院及广州市第八人民医院2015年3月至2016年2月的门诊或住院患者,其中男21例,女5例。所有病例的诊断符合2012年《肝衰竭诊治指南》的诊断标准。另对照组中包括16例CHB患者,男15例,女1例;代偿性肝硬化(liver cirrhosis,LC)患者17例,男15例,女2例;14例在院进行健康体检(health control,HC)各型肝炎血清学标志物阴性及肝功能正常的人群作为健康对照组。CHB及LC的诊断均符合2015年《慢性乙型肝炎防治指南》的诊断标准。入选的研究对象均排除以下情况:合并感染甲型肝炎病毒(hepatitis a virus,HAV),丙型肝炎病毒(hepatitis c virus,HCV),丁型肝炎病毒(hepatitis d virus,HDV),戊型肝炎病毒(hepatitis e virus,HEV)或人类免疫缺陷病毒(human immunodeficiency virus,HIV);合并糖尿病、甲状腺功能亢进、自身免疫性肝病等自身免疫性疾病;合并恶性肿瘤及其他严重代谢性疾病。所有研究对象均签署知情同意书。1.血浆中抗SLA水平采用ELISA方法检测HC、CHB、LC、H-A组患者血浆中的抗SLA的OD值。并且将该值与肝功能相关指标进行相关性分析。2.外周血Tfr细胞频数及相关表型分子表达情况利用流式细胞染色技术,检测慢性HBV感染者外周血Tfr细胞频数及表型分子GITR、CTLA-4的表达情况。比较各组间Tfr细胞频数及表型差异。3.血浆中相关细胞因子水平采用流式多因子检测(CBA)方法,检测各组血浆中的相关细胞因子(IFN-γ、TNF-α、IL-10、GranzymeA、GranzymeB)水平。比较各组及 H-A 组内的细胞因子浓度大小及平均荧光密度(MFI)。同时将HBV感染者血浆中相关细胞因子及抗SLA的OD值与Tfr细胞频数进行相关性分析。4.外周血Tfr细胞分泌细胞因子水平利用流式细胞内因子染色(ICS)技术,检测慢性HBV感染者外周血Tfr细胞在CD3和CD28刺激情况下,分泌细胞因子(GranzymeA、GranzymeB、IL-10、IL-21 和 TNF-α 及 IFN-γ)水平。5.统计分析计量资料以均数±标准差(Mean±SD)表示,两组间计量资料间采用两独立样本t检验,不满足方差分析条件采用Mann-Whitney U test及Spearman进行相关性分析。所有数据采用Graphpad Prism 5.0及SPSS 20.0进行处理及作图。所有的统计分析基于双侧假设检验,以α =0.05为检验水准,P0.05有统计学意义。结果1.慢性HBV感染组血浆中的抗SLA水平均显著高于HC组(P0.001),而H-A组抗SLA的OD值显著高于CHB(P0.001)、LC组(P0.001);在HBV感染者中,抗SLA的OD值大小与ALT(R=0.260,P=0.046)及TBIL(R=0.646,P0.001)呈正相关,与 HBV-DNA 无明显相关(R=-0.069,P=0.598)。在H-A组,抗SLA的OD值大小与总胆酸(TBA)呈正相关(R=0.478,P=0.024),与凝血酶原活动度(PTA)、ALT、AST、DBIL、TBIL无显著相关性。2.慢性HBV感染者外周血CD4+T细胞频数无显著性差异,H-A组中的Tfr 细胞频数明显低于 HC(P0.001)、CHB(P0.001)、LC(P0.001),H-A组中的 Tfh 细胞频数明显低于 HC(P0.001)、CHB(P0.010)、LC(P0.001),且在 H-A 组中的 Tfr/Tfh 明显低于 HC(P0.001)、CHB(P0.001)、LC(P0.001)。H-A患者外周血中Tfr细胞的两种主要功能表型(GITR和CTLA-4),CTLA-4的表达明显高于GITR(P0.050),但是HBV感染者外周血中Tfr细胞表达两种表型无显著差异。3.H-A患者血浆中的2种细胞因子(IFN-γ、TNF-α)浓度明显高于CHB(P0.050,P0.001),GranzymeA 明显高于 HC(P0.010)。而 IL-10 浓度明显高于 HC(P0.001)、CHB(P0.050)、LC(P0.001)。而 GranzymeB 的水平没有显著差异。在H-A组内的5种细胞因子浓度无显著差异。H-A血浆中分泌的4种细胞因子(IFN-γ、TNF-α、IL-10、GranzymeA)MFI明显高于HC、CHB、或者LC,并且H-A组内血浆中5种细胞因子间的MFI均存在显著差异。4.慢性HBV感染者中Tfr细胞频数与血浆中IL-10水平呈负相关(R=-0.390,P=0.030),同时与血浆中分泌的抗SLA呈负相关(R=-0.450,P0.010),而与 GranzymeA 及 GranzymeB 无显著相关。5.慢性HBV感染者PBMC经CD3/CD28刺激后,Tfr细胞相关的6种细胞因子(GranzymeA、GranzymeB、IL-10、IL-21、TNF-α 及 IFN-γ)无显著差异。H-A组内CD3/CD28刺激后,Tfr细胞相关的6种细胞因子(GranzymeA、GranzymeB、IL-10、IL-21、TNF-α 及 IFN-γ)也无显著差异。结论H-A患者的抗SLA水平表达显著增加,提示了在肝衰竭状态下患者体液免疫环境发生了变化。H-A患者Tfr细胞频数与抗SLA水平负相关,同时与相关细胞因子(IL-10)呈负相关,均提示了 Tfr细胞在H-A患者高表达SLA中发挥了重要作用。未来的体外研究将进一步验证这一结论。
[Abstract]:Background: liver failure is a serious liver disease, a common clinical symptom severity, complications, treatment difficulties, in our country the mortality rate is up to 60 ~ 80%. in China cause liver failure mainly for hepatitis B virus (hepatitis B, virus, HBV) infection, the main clinical manifestations in acute on chronic liver failure (acute-on-chronic liver failure, ACLF). In China, acute liver failure (acute liver failure, AHF) and subacute liver failure showed a decreasing trend, and acute on chronic liver failure and chronic liver failure increased.HBV related acute on chronic liver failure (HBV-related acute-on-chronic liver failure, H-A) in the pathogenesis, body specific HBV immunity and non-specific immune status, compared with other chronic HBV infection status, has undergone great changes. The humoral immunity has experienced many changes, such as in patients with H-A The surface antigen (Hepatitis B virus surface antigen, HBsAg) level in patients with chronic hepatitis B (chronic hepatitis, B, CHB) is low, and the conversion of serum anti HBe HBeAg/, even in many patients will autoantibodies positive situation. In the study of chronic HBV infection in the process of the emergence of a large number of their own antibody positive, including antinuclear antibody, anti mitochondrial antibody, anti smooth muscle antibody. The anti soluble liver antigen (anti-soluble liver antigen antibody anti-SLA/, anti SLA) is one of the types of liver antigens, studies have shown that anti SLA in CHB, compensated cirrhosis, decompensated cirrhosis, liver cell cancer (hepatocellular carcinoma, HCC), acute liver failure (acute liver failure, AHF) positive rate were 2.2%, 3.5%, 1.7%, 2.6%, 21.9%.. However, there is no research on the anti SLA in H-A, and And why patients with liver failure in autoantibody expression, its mechanism is not clear. The production of antibodies is a delicate and complicated process. T follicular helper cells (Follicular T helper cells, Tfh cells) B cell differentiation into plasma cells that produce antibodies, and antibody class switching. The interaction between the two, play an important role in the humoral immune response. Studies show that in autoimmune diseases, the frequency of Tfh cells and its subsets changes, and B cell in a dysfunctional state. In the germinal center (Germinal Center GC) follicular regulatory T cells (FollicularTregulatory, cells, Tfr cells), mainly involved in the germinal center the reaction and inhibition of Tfh cells and B cells, to avoid the production of autoantibodies associated with autoimmune diseases. Therefore, this paper aims to study the effects of Tfr cells on patients with anti SLA H-A abnormal expression. The purpose of this study is to HB Transverse V infection among natural history as the research object, the degree of expression of SLA autoantibodies mainly study H-A patients, and the relationship between high expression of anti SLA and Tfr cell frequency and function. Methods 1. subjects of 26 cases with H-A in our hospital and the Eighth People's Hospital of Guangzhou from March 2015 to February 2016 in the outpatient or hospitalized patients, including 21 male cases, 5 cases were female. The diagnosis of all cases meet the diagnostic criteria of 2012 "guide for diagnosis and treatment of liver failure. Other > control group including 16 cases of CHB patients, male 15 cases, female 1 cases; decompensated cirrhosis (liver cirrhosis, LC) in 17 patients, male 15 cases, female 2 cases; 14 cases health examination in the hospital (health, control, HC) hepatitis B serological markers were negative and normal liver function group as the control group and health diagnosis.CHB LC were consistent with the diagnostic criteria of 2015" chronic hepatitis B Prevention Guide >. Eligible studies were excluded from the following situations: Co infection of hepatitis A virus (hepatitis A, virus, HAV), hepatitis C virus (hepatitis C, virus, HCV), hepatitis D virus (hepatitis D, virus, HDV), hepatitis E virus (hepatitis E, virus, HEV) or human immunodeficiency virus (human immunodeficiency, virus, HIV); diabetes, hyperthyroidism. Autoimmune liver disease and other autoimmune diseases; malignant tumor and other serious metabolic diseases. All subjects were informed consent of anti SLA level.1. in plasma by ELISA method for detection of HC, CHB, LC, H-A in plasma in patients with anti SLA. And the OD value is associated with liver function analyzed the expression by flow cytometry staining of.2. in peripheral blood Tfr cells and related molecular phenotype frequency, detection of chronic HBV infection of Tfr cells in peripheral blood and the frequency of the phenotype GITR, CTLA-4 expression. Conditions were compared between the groups. Correlation between cytokine levels and phenotypic differences in the frequency of Tfr cells.3. in plasma by flow cytometry (CBA) method for detection of multi factor and related cytokines were detected in plasma (IFN- gamma, alpha TNF-, IL-10, GranzymeA, GranzymeB and H-A) levels were compared between the groups. In the group of cytokines concentration and size the mean fluorescence intensity (MFI). At the same time, OD HBV infection related cytokines in plasma and the anti SLA and Tfr cell frequency correlation analysis of cytokine secretion by flow cytometry in factor Tfr staining cells of peripheral blood.4. (ICS) technology, Tfr cells in the peripheral blood of patients with chronic HBV infection detection in CD3 and CD28 stimulation of the secretion of cytokines (GranzymeA, GranzymeB, IL-10, IL-21 and TNF- alpha and IFN- gamma) level.5. statistical analysis of measurement data to mean + standard deviation (Mean + SD) said that the measurement data between the two groups using two independent samples t Test and analysis of variance does not meet the conditions with the Mann-Whitney U test and Spearman correlation analysis. All data using Graphpad Prism 5 and SPSS 20 for processing and mapping. All statistical analysis based on bilateral hypothesis test, to test the level of a =0.05, there was statistical significance P0.05. Results 1. chronic HBV infection group in plasma anti SLA the level is significantly higher than the HC group (P0.001), and H-A group of anti SLA OD value was significantly higher than that of CHB (P0.001), LC group (P0.001); in HBV infection, anti SLA and ALT od size (R=0.260, P=0.046) and TBIL (R=0.646, P0.001) were positively correlated with no obvious correlation (HBV-DNA R=-0.069, P=0.598). In group H-A, the OD values of anti SLA and total bile acid (TBA) was positively correlated (R=0.478, P=0.024), and prothrombin activity (PTA), ALT, AST, DBIL, TBIL and.2. showed no significant correlation with chronic HBV infection of peripheral blood CD4+T cells had no significant difference of frequency 寮，

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