本文關(guān)鍵詞： HBV相關(guān)慢加性急肝衰竭 濾泡調(diào)節(jié)性T細(xì)胞 肝抗原自身抗體 自身免疫　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景肝衰竭是臨床常見(jiàn)的嚴(yán)重肝病癥候群,病情嚴(yán)重、并發(fā)癥多、治療困難,在我國(guó)病死率高達(dá)60～80%。我國(guó)肝衰竭的病因主要為乙型肝炎病毒(hepatitis B virus,HBV)感染,目前臨床表現(xiàn)主要以慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)為主。在我國(guó)急性肝衰竭(acute liver failure,AHF)和亞急性肝衰竭呈現(xiàn)減少趨勢(shì),而慢加急性肝衰竭和慢性肝衰竭呈增加趨勢(shì)。HBV 相關(guān)慢加急性肝衰竭(HBV-related acute-on-chronic liver failure,H-A)的發(fā)病過(guò)程中,機(jī)體針對(duì)HBV的特異性免疫和非特異性免疫狀態(tài),與其他慢性HBV感染狀態(tài)相比,發(fā)生了很大的變化。其中機(jī)體體液免疫方面也發(fā)生了很多的變化,如在H-A患者體內(nèi)其表面抗原(Hepatitis B virus surface antigen,HBsAg)水平較慢性乙型肝炎(chronic hepatitis B,CHB)低,并且發(fā)生了 HBeAg/抗HBe的血清學(xué)轉(zhuǎn)換,甚至在很多患者體內(nèi)會(huì)出現(xiàn)自身抗體譜轉(zhuǎn)陽(yáng)的情況。研究中發(fā)現(xiàn)在慢性HBV感染過(guò)程中出現(xiàn)了大量自身抗體的轉(zhuǎn)陽(yáng),包括抗核抗體、抗線粒體抗體、抗平滑肌抗體等。其中抗可溶性肝抗原抗體(anti-soluble liver antigen antibody,anti-SLA/抗SLA)是肝抗原自身抗體的類型之一,有研究表明抗SLA在CHB、代償性肝硬化、非代償性肝硬化、肝細(xì)胞癌(hepatocellular carcinoma,HCC)、急性肝衰竭(acute liver failure,AHF)中的陽(yáng)性率分別為2.2%、3.5%、1.7%、2.6%、21.9%。但是,目前還沒(méi)有關(guān)于抗SLA在H-A中的研究,而且為什么肝衰竭的患者中自身抗體表達(dá)上調(diào),其機(jī)理也不清楚�？贵w的產(chǎn)生是精細(xì)而復(fù)雜的過(guò)程。濾泡輔助性T細(xì)胞(Follicular T helper cells,Tfh細(xì)胞)輔助B細(xì)胞分化為漿細(xì)胞、產(chǎn)生抗體、及抗體類別轉(zhuǎn)換。二者相互作用,在體液免疫應(yīng)答中發(fā)揮重要作用。研究顯示在自身免疫疾病中,Tfh細(xì)胞及其亞群頻數(shù)發(fā)生變化,同時(shí)B細(xì)胞處于功能失調(diào)狀態(tài)。在生發(fā)中心(Germinal Center,GC)的濾泡調(diào)節(jié)性 T 細(xì)胞(FollicularTregulatory cells,Tfr細(xì)胞),主要參與生發(fā)中心的反應(yīng),抑制Tfh細(xì)胞和B細(xì)胞,避免自身抗體的產(chǎn)生,與自身免疫性疾病相關(guān)。因此本文旨在研究Tfr細(xì)胞對(duì)H-A患者血漿抗SLA異常表達(dá)的影響。目的本研究是以HBV感染自然史的橫向人群為研究對(duì)象,主要研究H-A患者表達(dá)自身抗體SLA的程度,以及高表達(dá)抗SLA與Tfr細(xì)胞頻數(shù)和功能的關(guān)系。方法1.研究對(duì)象26例H-A為本院及廣州市第八人民醫(yī)院2015年3月至2016年2月的門診或住院患者,其中男21例,女5例。所有病例的診斷符合2012年《肝衰竭診治指南》的診斷標(biāo)準(zhǔn)。另對(duì)照組中包括16例CHB患者,男15例,女1例;代償性肝硬化(liver cirrhosis,LC)患者17例,男15例,女2例;14例在院進(jìn)行健康體檢(health control,HC)各型肝炎血清學(xué)標(biāo)志物陰性及肝功能正常的人群作為健康對(duì)照組。CHB及LC的診斷均符合2015年《慢性乙型肝炎防治指南》的診斷標(biāo)準(zhǔn)。入選的研究對(duì)象均排除以下情況: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)或人類免疫缺陷病毒(human immunodeficiency virus,HIV);合并糖尿病、甲狀腺功能亢進(jìn)、自身免疫性肝病等自身免疫性疾病;合并惡性腫瘤及其他嚴(yán)重代謝性疾病。所有研究對(duì)象均簽署知情同意書(shū)。1.血漿中抗SLA水平采用ELISA方法檢測(cè)HC、CHB、LC、H-A組患者血漿中的抗SLA的OD值。并且將該值與肝功能相關(guān)指標(biāo)進(jìn)行相關(guān)性分析。2.外周血Tfr細(xì)胞頻數(shù)及相關(guān)表型分子表達(dá)情況利用流式細(xì)胞染色技術(shù),檢測(cè)慢性HBV感染者外周血Tfr細(xì)胞頻數(shù)及表型分子GITR、CTLA-4的表達(dá)情況。比較各組間Tfr細(xì)胞頻數(shù)及表型差異。3.血漿中相關(guān)細(xì)胞因子水平采用流式多因子檢測(cè)(CBA)方法,檢測(cè)各組血漿中的相關(guān)細(xì)胞因子(IFN-γ、TNF-α、IL-10、GranzymeA、GranzymeB)水平。比較各組及 H-A 組內(nèi)的細(xì)胞因子濃度大小及平均熒光密度(MFI)。同時(shí)將HBV感染者血漿中相關(guān)細(xì)胞因子及抗SLA的OD值與Tfr細(xì)胞頻數(shù)進(jìn)行相關(guān)性分析。4.外周血Tfr細(xì)胞分泌細(xì)胞因子水平利用流式細(xì)胞內(nèi)因子染色(ICS)技術(shù),檢測(cè)慢性HBV感染者外周血Tfr細(xì)胞在CD3和CD28刺激情況下,分泌細(xì)胞因子(GranzymeA、GranzymeB、IL-10、IL-21 和 TNF-α 及 IFN-γ)水平。5.統(tǒng)計(jì)分析計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示,兩組間計(jì)量資料間采用兩獨(dú)立樣本t檢驗(yàn),不滿足方差分析條件采用Mann-Whitney U test及Spearman進(jìn)行相關(guān)性分析。所有數(shù)據(jù)采用Graphpad Prism 5.0及SPSS 20.0進(jìn)行處理及作圖。所有的統(tǒng)計(jì)分析基于雙側(cè)假設(shè)檢驗(yàn),以α =0.05為檢驗(yàn)水準(zhǔn),P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果1.慢性HBV感染組血漿中的抗SLA水平均顯著高于HC組(P0.001),而H-A組抗SLA的OD值顯著高于CHB(P0.001)、LC組(P0.001);在HBV感染者中,抗SLA的OD值大小與ALT(R=0.260,P=0.046)及TBIL(R=0.646,P0.001)呈正相關(guān),與 HBV-DNA 無(wú)明顯相關(guān)(R=-0.069,P=0.598)。在H-A組,抗SLA的OD值大小與總膽酸(TBA)呈正相關(guān)(R=0.478,P=0.024),與凝血酶原活動(dòng)度(PTA)、ALT、AST、DBIL、TBIL無(wú)顯著相關(guān)性。2.慢性HBV感染者外周血CD4+T細(xì)胞頻數(shù)無(wú)顯著性差異,H-A組中的Tfr 細(xì)胞頻數(shù)明顯低于 HC(P0.001)、CHB(P0.001)、LC(P0.001),H-A組中的 Tfh 細(xì)胞頻數(shù)明顯低于 HC(P0.001)、CHB(P0.010)、LC(P0.001),且在 H-A 組中的 Tfr/Tfh 明顯低于 HC(P0.001)、CHB(P0.001)、LC(P0.001)。H-A患者外周血中Tfr細(xì)胞的兩種主要功能表型(GITR和CTLA-4),CTLA-4的表達(dá)明顯高于GITR(P0.050),但是HBV感染者外周血中Tfr細(xì)胞表達(dá)兩種表型無(wú)顯著差異。3.H-A患者血漿中的2種細(xì)胞因子(IFN-γ、TNF-α)濃度明顯高于CHB(P0.050,P0.001),GranzymeA 明顯高于 HC(P0.010)。而 IL-10 濃度明顯高于 HC(P0.001)、CHB(P0.050)、LC(P0.001)。而 GranzymeB 的水平?jīng)]有顯著差異。在H-A組內(nèi)的5種細(xì)胞因子濃度無(wú)顯著差異。H-A血漿中分泌的4種細(xì)胞因子(IFN-γ、TNF-α、IL-10、GranzymeA)MFI明顯高于HC、CHB、或者LC,并且H-A組內(nèi)血漿中5種細(xì)胞因子間的MFI均存在顯著差異。4.慢性HBV感染者中Tfr細(xì)胞頻數(shù)與血漿中IL-10水平呈負(fù)相關(guān)(R=-0.390,P=0.030),同時(shí)與血漿中分泌的抗SLA呈負(fù)相關(guān)(R=-0.450,P0.010),而與 GranzymeA 及 GranzymeB 無(wú)顯著相關(guān)。5.慢性HBV感染者PBMC經(jīng)CD3/CD28刺激后,Tfr細(xì)胞相關(guān)的6種細(xì)胞因子(GranzymeA、GranzymeB、IL-10、IL-21、TNF-α 及 IFN-γ)無(wú)顯著差異。H-A組內(nèi)CD3/CD28刺激后,Tfr細(xì)胞相關(guān)的6種細(xì)胞因子(GranzymeA、GranzymeB、IL-10、IL-21、TNF-α 及 IFN-γ)也無(wú)顯著差異。結(jié)論H-A患者的抗SLA水平表達(dá)顯著增加,提示了在肝衰竭狀態(tài)下患者體液免疫環(huán)境發(fā)生了變化。H-A患者Tfr細(xì)胞頻數(shù)與抗SLA水平負(fù)相關(guān),同時(shí)與相關(guān)細(xì)胞因子(IL-10)呈負(fù)相關(guān),均提示了 Tfr細(xì)胞在H-A患者高表達(dá)SLA中發(fā)揮了重要作用。未來(lái)的體外研究將進(jìn)一步驗(yàn)證這一結(jié)論。
[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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