本文選題：慢加急性乙型肝炎肝衰竭 + 慢性乙型肝炎�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：第一部分：慢加急性乙型肝炎肝衰竭患者BATF高表達(dá)與Th17免疫反應(yīng)增強的相關(guān)性研究研究背景 乙型肝炎病毒(hepatitis B virus, HBV)是一種嗜肝的非細(xì)胞毒性DNA病毒。目前全球約有3.5億至4億慢性HBV感染者,其中部分患者在促發(fā)因素的作用下,病情急性加重,迅速進(jìn)展為慢加急性乙型肝炎肝衰竭(acute-on-chronic hepatitis B liver failure, ACHBLF). ACHBLF是指在慢性HBV感染的基礎(chǔ)上,肝功能急劇、嚴(yán)重惡化,患者臨床表現(xiàn)以黃疸和凝血障礙為主,并于4周內(nèi)出現(xiàn)腹水和/或肝性腦病。宿主和病毒雙重因素的協(xié)同作用決定ACHBLF的疾病進(jìn)展,其中免疫因素逐漸被重視,在ACHBLF的疾病過程中起到關(guān)鍵作用。我們的前期研究檢測了輔助性T淋巴細(xì)胞17(Th17)細(xì)胞亞群在ACHBLF發(fā)生發(fā)展中的變化規(guī)律,發(fā)現(xiàn)ACHBLF患者Th17細(xì)胞亞群比例增加,其效應(yīng)細(xì)胞因子白細(xì)胞介素-17(IL-17)增多,Th17免疫反應(yīng)增強與患者病情進(jìn)展和預(yù)后明顯相關(guān)。我們進(jìn)一步探討了Th17細(xì)胞增多的可能原因,發(fā)現(xiàn)堿性亮氨酸拉鏈轉(zhuǎn)錄因子ATF樣蛋白(basic leucine zipper transcription factor, ATF-like, BATF)可能是調(diào)節(jié)慢加急性乙型肝炎肝衰竭Th17免疫反應(yīng)的分子靶標(biāo)。BATF屬于激活蛋白-1轉(zhuǎn)錄因子超家族,在多種免疫細(xì)胞的分化發(fā)育和免疫球蛋白類別轉(zhuǎn)換中發(fā)揮了重要作用。最近的研究發(fā)現(xiàn),BATF是調(diào)控Thl7細(xì)胞增殖分化和IL-17產(chǎn)生的關(guān)鍵因素,介導(dǎo)疾病免疫損傷。然而,ACHBLF患者體內(nèi)BATF表達(dá)的變化及其與Thl7免疫應(yīng)答的關(guān)系尚不清楚。 研究目的 1.探討ACHBLF患者外周血BATF基因和蛋白表達(dá)水平的變化； 2.探討ACHBLF患者BATF表達(dá)水平與肝臟炎癥損傷程度以及Th17免疫應(yīng)答 的關(guān)系。研究方法本研究共納入22例ACHBLF患者,60例慢性乙型肝炎(chronic hepatitis B,CHB)患者以及17例健康對照作為研究對象。應(yīng)用熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(real-time polymerase chain reaction, RT-PCR)方法檢測ACHBLF患者、CHB患者及健康對照外周血單個核細(xì)胞(peripheral blood mononuclear cells, PBMCs)中BATF及IL-17、IFN-γ、RORyt和T-bet mRNA表達(dá)水平；采用流式細(xì)胞術(shù)檢測外周血CD3+BATF+T細(xì)胞及Thl、Th17和Tc1細(xì)胞比例。另外收集了26例行經(jīng)皮肝穿刺活檢術(shù)的CHB患者及6例健康肝移植供者的肝組織,應(yīng)用免疫組化方法檢測BATF在肝組織內(nèi)的表達(dá)及定位。研究結(jié)果 1. ACHBLF患者PBMCs中BATF mRNA表達(dá)明顯高于健康對照和CHB患者(P0.01, P0.05), CHB患者BATF mRNA表達(dá)較健康對照也明顯增高(P0.01)。 2. ACHBLF患者PBMCs中BATF mRNA水平與患者總膽紅素水平呈正相關(guān)(r=0.477,P=0.025); CHB患者BATF mRNA水平與患者谷丙轉(zhuǎn)氨酶水平、HBV-DNA病毒量呈正相關(guān)(r=0.497, P0.001; r=0.340, P=0,008). 3. ACHBLF患者外周血CD3+BATF+細(xì)胞比例明顯高于健康對照和CHB患者(P0.01, P0.01), CHB患者CD3+BATF+細(xì)胞比例也較健康對照增高(P0.01)。三組研究對象CD3+T細(xì)胞中BATF蛋白平均熒光強度也表現(xiàn)出相同的變化趨勢(P0.01,P0.05,P0.01)。 4. ACHBLF患者和CHB患者外周血CD3+BATF+T細(xì)胞比例與Th17細(xì)胞比例呈正相關(guān)(r=0.338,P=0.002)；外周血單個核細(xì)胞中Th17細(xì)胞效應(yīng)細(xì)胞因子IL-17及轉(zhuǎn)錄因子RORγt mRNA水平也與CD3+BATF+T細(xì)胞比例呈明顯正相關(guān)(r=0.303,P=0.006;r=0.256,P=0.020)。 5. BATF陽性細(xì)胞在CHB患者肝臟門脈區(qū)聚集。與健康對照相比,CHB患者肝臟浸潤BATF陽性細(xì)胞明顯增多(P0.01),并且BATF陽性細(xì)胞數(shù)目隨肝臟炎癥程度增高而增多(P0.05)。另外,11例應(yīng)用抗病毒藥物治療6個月后出現(xiàn)生物學(xué)應(yīng)答和病毒性應(yīng)答的CHB患者中,治療后外周血單個核細(xì) 胞BATF mRNA水平較治療前明顯降低(P=0.01)。結(jié)論 ACHBLF患者、CHB患者外周血和肝組織內(nèi)BATF表達(dá)明顯增高,并且與肝臟炎癥損傷程度相關(guān),提示BATF在HBV感染慢性化和重癥化中均發(fā)揮作用。患者BATF表達(dá)水平與Th17免疫應(yīng)答強度正相關(guān),BATF可能通過上調(diào)Th17免疫反應(yīng),參與介導(dǎo)肝臟免疫損傷。 第二部分：TIPE2在慢加急性乙型肝炎肝衰竭患者外周血單個核細(xì)胞中的表達(dá)及其與疾病預(yù)后的關(guān)系 研究背景 免疫負(fù)調(diào)控分子表達(dá)異常是ACHBLF免疫穩(wěn)態(tài)失衡的另一重要原因。越來越多的研究發(fā)現(xiàn)ACHBLF患者出現(xiàn)“免疫麻痹”。這種免疫抑制由多種免疫調(diào)節(jié)分子和調(diào)節(jié)性免疫細(xì)胞參與介導(dǎo),可能是機體對早期過度促炎癥反應(yīng)的拮抗效應(yīng)。腫瘤壞死因子-α誘導(dǎo)蛋白8型-2(tumor necrosis factor-α-induced protein8-like2,TNFAIP8L-2,TIPE2)是最近發(fā)現(xiàn)的一種免疫調(diào)控分子,主要表達(dá)于免疫器官和淋巴組織中,不同發(fā)育階段的巨噬細(xì)胞、T細(xì)胞和B細(xì)胞中均有TIPE2表達(dá)。TIPE2基因缺陷細(xì)胞表現(xiàn)出對T細(xì)胞受體和Toll樣受體信號活化的高反應(yīng)性,說明TIPE2可以負(fù)性調(diào)節(jié)固有免疫和適應(yīng)性免疫反應(yīng)。TIPE2的正常表達(dá)是阻止免疫反應(yīng)過激和維持免疫穩(wěn)態(tài)所必需的。研究發(fā)現(xiàn),TIPE2在系統(tǒng)性紅斑狼瘡和慢性乙型肝炎等免疫炎癥疾病中具有重要作用。TIPE2參與HBV引起的肝臟免疫損傷,但TIPE2在ACHBLF患者中的表達(dá)改變及意義目前尚無研究。 研究目的 1.探討ACHBLF患者外周血單個核細(xì)胞中TIPE2mRNA的表達(dá)改變,以及TIPE2與ACHBLF病情嚴(yán)重程度和疾病預(yù)后的關(guān)系； 2.初步探討TIPE2影響ACHBLF病情發(fā)展的作用機制。研究方法本研究共納入56例ACHBLF患者,60例CHB患者和24例健康對照者。我們對ACHBLF患者進(jìn)行了3個月隨訪,根據(jù)患者3個月后的生存情況將患者分為存活組和死亡組。通過密度梯度離心法提取研究對象外周血單個核細(xì)胞,Trizol提取總RNA,采用RT-PCR法檢測PBMCs中TIPE2mRNA的相對表達(dá)水平。另外,體外培養(yǎng)外周血單個核細(xì)胞,檢測應(yīng)用脂多糖(lipopolysaccharide, LPS)刺激后單個核細(xì)胞中TIPE2、IL-6和TNF-α mRNA的表達(dá)水平。促炎細(xì)胞因子IL-6和TNF-α是單個核細(xì)胞分泌的主要細(xì)胞因子,其體外分泌水平被認(rèn)為可以代表機體細(xì)胞免疫水平。 研究結(jié)果 1. ACHBLF患者PBMCs中TIPE2mRNA水平明顯高于健康對照組(P0.05)和CHB患者(P0.01)。與健康對照相比,CHB患者TIPE2mRNA水平降低(P0.05)。 2. ACHBLF患者TIPE2mRNA表達(dá)水平與患者總膽紅素水平(r=0.300,P=0.024)、凝血酶原時間的國際標(biāo)準(zhǔn)化比值(r=0.301,P=0.024)和終末期肝病模型積分(r=0.335,P=0.011)呈明顯正相關(guān)關(guān)系,與谷丙轉(zhuǎn)氨酶、白蛋白、肌酐水平、凝血酶原活動度、HBV-DNA病毒量和HBeAg均不具有相關(guān)性。 3. ACHBLF死亡組患者TIPE2表達(dá)明顯高于存活組患者(P0.01)。用TIPE2預(yù)測ACHBLF患者預(yù)后,其受試者工作特征曲線下面積為0.721(95%CI0.572-0.870)。我們進(jìn)一步研究了ACHBLF患者TIPE2表達(dá)的動態(tài)改變。收集32例ACHBLF患者入院第二天、第一周、第二周和第三周的連續(xù)血樣標(biāo)本,研究發(fā)現(xiàn),18例存活患者入院三周內(nèi)PBMCs TIPE2表達(dá)隨病情好轉(zhuǎn)降低,而14例死亡患者TIPE2表達(dá)不降反而升高。 4. TIPE2與ACHBLF患者細(xì)胞免疫功能下降有關(guān)。與健康對照和CHB患者相比,ACHBLF患者體外刺激的單個核細(xì)胞TIPE2mRNA表達(dá)水平明顯增高(P均0.05),相反,IL-6和TNF-a mRNA的表達(dá)水平明顯降低(P均0.05),在死亡組ACHBLF患者中這種趨勢更為明顯。另外,LPS刺激后外周血單個核細(xì)胞中TIPE2與TNF-α的表達(dá)呈負(fù)相關(guān)(r=-0.337,P=0.048)。 結(jié)論： TIPE2在慢性HBV感染重癥化過程中發(fā)揮重要作用。ACHBLF患者TIPE2基因表達(dá)水平與病情嚴(yán)重程度正相關(guān),TIPE2基因表達(dá)增高是患者預(yù)后不良的預(yù)測指標(biāo)。TIPE2可能通過負(fù)性調(diào)控細(xì)胞免疫反應(yīng)介導(dǎo)ACHBLF的發(fā)生和發(fā)展,這為探尋新的ACHBLF治療靶點提供了基礎(chǔ)。
[Abstract]:Part I : Background of Study on the Relationship between High Expression of BATF and Th17 Immune Response in Patients with Chronic Hepatitis B Hepatic Failure

Hepatitis B virus ( HBV ) is a non - cytotoxic DNA virus of liver . At present , there are about 350 million to 400 million chronic HBV infection in the world . Some of these patients have acute exacerbation of acute hepatitis B ( acute - on - chronic hepatitis B liver failure , ACHBLF ) . In this study , we have studied the changes of Th17 cell subsets in the development of ACHBLF , and found that the basic leucine zipper transcription factor ATF - like ( BATF ) plays a key role in the pathogenesis of ACHBLF .

Purpose of study

1 . To investigate the changes of BATF gene and protein expression in peripheral blood of patients with ACHBLF ;


2 . To investigate the level of BATF expression in patients with ACHBLF and the degree of liver inflammation and Th17 immune response

Methods The expression of BATF and IL - 17 , IFN - 緯 , RORyt and T - bet mRNA in peripheral blood mononuclear cells ( PBMCs ) of 22 patients with ACHBLF , 60 patients with chronic hepatitis B , and 17 healthy controls were studied by real - time polymerase chain reaction ( RT - PCR ) .
The proportions of CD3 + BATF + T cells and Thl , Th17 and Tc1 cells in peripheral blood were detected by flow cytometry .

1 . The expression of BATF mRNA in PBMCs of patients with ACHBLF was significantly higher than that in healthy controls ( P0.01 ) .

2 . The level of BATF mRNA in PBMCs of patients with ACHBLF was positively correlated with the level of total bilirubin ( r = 0.477 , P = 0.025 ) . The level of BATF mRNA was positively correlated with the level of alanine aminotransferase and HBV - DNA virus ( r = 0.497 , P0.001 ; r = 0.340 , P = 0.008 ) .

3 . The percentage of CD3 + BATF + cells in peripheral blood of patients with ACHBLF was significantly higher than that in healthy controls ( P0.01 ) .

4 . The proportion of CD3 + BATF + T cells in peripheral blood of patients with ACHBLF was positively correlated with that of Th17 cells ( r = 0.338 , P = 0.002 ) .
The mRNA levels of Th17 cell effector cytokines , IL - 17 , and the transcription factor R - 緯 t mRNA in peripheral blood mononuclear cells were positively correlated with the ratio of CD3 + BATF + T cells ( r = 0.303 , P = 0.006 ; r = 0.256 , P = 0.020 ) .

5 . The positive cells of BATF positive cells were collected in the portal vein region of the liver of the patients . Compared with the healthy control , the number of BATF - positive cells increased significantly ( P0.01 ) , and the number of BATF - positive cells increased with the increase of the degree of inflammation of the liver ( P0.05 ) .

The level of BATF mRNA was significantly lower than that before treatment ( P = 0.01 ) . Conclusion

In patients with ACHBLF , the expression of BATF in peripheral blood and liver tissues increased significantly in patients with ACHBLF and correlated with the degree of liver inflammation , suggesting that BATF plays a role in both chronic and severe HBV infection . The level of BATF expression is positively related to the intensity of Th17 immune response , and BATF may be involved in mediating hepatic immune injury by up - regulation of Th17 immune response .

The second part : the expression of tipe2 in peripheral blood mononuclear cells of patients with chronic hepatitis B liver failure and its relationship with prognosis

Background of the study

The expression of immune negative regulatory molecules is another important reason for the imbalance of immune homeostasis in ACHBLF . More and more studies have found that there is an " immune paralysis " in patients with ACHBLF .

Purpose of study

1 . To investigate the expression of TIPE2mRNA in peripheral blood mononuclear cells ( PBMC ) of patients with ACHBLF , and to investigate the relationship between the severity of TIPE2 and ACHBLF and the prognosis of disease .


2 . A preliminary study was conducted to investigate the effect mechanism of TI2 on the development of ACHBLF . The study included 56 patients with ACHBLF , 60 patients with hepatitis B , and 24 healthy controls . We divided the patients into survival group and death group according to the survival of patients after 3 months .

Results of the study

1 . The TIPE2mRNA levels in PBMCs of patients with ACHBLF were significantly higher than those in healthy controls ( P0.05 ) .

2 . The expression level of TIPE2mRNA in ACHBLF was positively correlated with the level of total bilirubin ( r = 0.300 , P = 0 . 024 ) , prothrombin time ( r = 0.301 , P = 0 . 024 ) and end - stage liver disease model ( r = 0.335 , P = 0 . 011 ) .

3 . Patients with ACHBLF were significantly higher than those in the survival group ( P0.01 ) . The prognosis of patients with ACHBLF was predicted . The area under the working characteristic curve of ACHBLF was 0.721 ( 95 % CI 0.572 - 0.870 ) .

4 . The expression level of TIPE2mRNA in peripheral blood mononuclear cells stimulated by ACHBLF increased significantly ( P < 0.05 ) , but the expression level of IL - 6 and TNF - a mRNA was significantly decreased ( P < 0.05 ) . In addition , the expression of TIR2 and TNF - 偽 in peripheral blood mononuclear cells was negatively correlated with LPS ( r = - 0.337 , P = 0.048 ) .

Conclusion :

The expression level of TI2 gene is closely related to the severity of the disease . The expression level of TI2 gene is associated with the severity of the disease . The expression of TI2 gene is the predictor of poor prognosis of patients .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.62;R575.3

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