BATF和TIPE2在慢加急性乙型肝炎肝衰竭患者中的表达及意义

发布时间：2018-05-02 20:18

本文选题：慢加急性乙型肝炎肝衰竭 + 慢性乙型肝炎　；参考：《山东大学》2014年博士论文

【摘要】：第一部分：慢加急性乙型肝炎肝衰竭患者BATF高表达与Th17免疫反应增强的相关性研究研究背景乙型肝炎病毒(hepatitis B virus, HBV)是一种嗜肝的非细胞毒性DNA病毒。目前全球约有3.5亿至4亿慢性HBV感染者,其中部分患者在促发因素的作用下,病情急性加重,迅速进展为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感染的基础上,肝功能急剧、严重恶化,患者临床表现以黄疸和凝血障碍为主,并于4周内出现腹水和/或肝性脑病。宿主和病毒双重因素的协同作用决定ACHBLF的疾病进展,其中免疫因素逐渐被重视,在ACHBLF的疾病过程中起到关键作用。我们的前期研究检测了辅助性T淋巴细胞17(Th17)细胞亚群在ACHBLF发生发展中的变化规律,发现ACHBLF患者Th17细胞亚群比例增加,其效应细胞因子白细胞介素-17(IL-17)增多,Th17免疫反应增强与患者病情进展和预后明显相关。我们进一步探讨了Th17细胞增多的可能原因,发现碱性亮氨酸拉链转录因子ATF样蛋白(basic leucine zipper transcription factor, ATF-like, BATF)可能是调节慢加急性乙型肝炎肝衰竭Th17免疫反应的分子靶标。BATF属于激活蛋白-1转录因子超家族,在多种免疫细胞的分化发育和免疫球蛋白类别转换中发挥了重要作用。最近的研究发现,BATF是调控Thl7细胞增殖分化和IL-17产生的关键因素,介导疾病免疫损伤。然而,ACHBLF患者体内BATF表达的变化及其与Thl7免疫应答的关系尚不清楚。研究目的 1.探讨ACHBLF患者外周血BATF基因和蛋白表达水平的变化； 2.探讨ACHBLF患者BATF表达水平与肝脏炎症损伤程度以及Th17免疫应答的关系。研究方法本研究共纳入22例ACHBLF患者,60例慢性乙型肝炎(chronic hepatitis B,CHB)患者以及17例健康对照作为研究对象。应用荧光定量聚合酶链式反应(real-time polymerase chain reaction, RT-PCR)方法检测ACHBLF患者、CHB患者及健康对照外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)中BATF及IL-17、IFN-γ、RORyt和T-bet mRNA表达水平；采用流式细胞术检测外周血CD3+BATF+T细胞及Thl、Th17和Tc1细胞比例。另外收集了26例行经皮肝穿刺活检术的CHB患者及6例健康肝移植供者的肝组织,应用免疫组化方法检测BATF在肝组织内的表达及定位。研究结果 1. ACHBLF患者PBMCs中BATF mRNA表达明显高于健康对照和CHB患者(P0.01, P0.05), CHB患者BATF mRNA表达较健康对照也明显增高(P0.01)。 2. ACHBLF患者PBMCs中BATF mRNA水平与患者总胆红素水平呈正相关(r=0.477,P=0.025); CHB患者BATF mRNA水平与患者谷丙转氨酶水平、HBV-DNA病毒量呈正相关(r=0.497, P0.001; r=0.340, P=0,008). 3. ACHBLF患者外周血CD3+BATF+细胞比例明显高于健康对照和CHB患者(P0.01, P0.01), CHB患者CD3+BATF+细胞比例也较健康对照增高(P0.01)。三组研究对象CD3+T细胞中BATF蛋白平均荧光强度也表现出相同的变化趋势(P0.01,P0.05,P0.01)。 4. ACHBLF患者和CHB患者外周血CD3+BATF+T细胞比例与Th17细胞比例呈正相关(r=0.338,P=0.002)；外周血单个核细胞中Th17细胞效应细胞因子IL-17及转录因子RORγt mRNA水平也与CD3+BATF+T细胞比例呈明显正相关(r=0.303,P=0.006;r=0.256,P=0.020)。 5. BATF阳性细胞在CHB患者肝脏门脉区聚集。与健康对照相比,CHB患者肝脏浸润BATF阳性细胞明显增多(P0.01),并且BATF阳性细胞数目随肝脏炎症程度增高而增多(P0.05)。另外,11例应用抗病毒药物治疗6个月后出现生物学应答和病毒性应答的CHB患者中,治疗后外周血单个核细胞BATF mRNA水平较治疗前明显降低(P=0.01)。结论 ACHBLF患者、CHB患者外周血和肝组织内BATF表达明显增高,并且与肝脏炎症损伤程度相关,提示BATF在HBV感染慢性化和重症化中均发挥作用。患者BATF表达水平与Th17免疫应答强度正相关,BATF可能通过上调Th17免疫反应,参与介导肝脏免疫损伤。第二部分：TIPE2在慢加急性乙型肝炎肝衰竭患者外周血单个核细胞中的表达及其与疾病预后的关系研究背景免疫负调控分子表达异常是ACHBLF免疫稳态失衡的另一重要原因。越来越多的研究发现ACHBLF患者出现“免疫麻痹”。这种免疫抑制由多种免疫调节分子和调节性免疫细胞参与介导,可能是机体对早期过度促炎症反应的拮抗效应。肿瘤坏死因子-α诱导蛋白8型-2(tumor necrosis factor-α-induced protein8-like2,TNFAIP8L-2,TIPE2)是最近发现的一种免疫调控分子,主要表达于免疫器官和淋巴组织中,不同发育阶段的巨噬细胞、T细胞和B细胞中均有TIPE2表达。TIPE2基因缺陷细胞表现出对T细胞受体和Toll样受体信号活化的高反应性,说明TIPE2可以负性调节固有免疫和适应性免疫反应。TIPE2的正常表达是阻止免疫反应过激和维持免疫稳态所必需的。研究发现,TIPE2在系统性红斑狼疮和慢性乙型肝炎等免疫炎症疾病中具有重要作用。TIPE2参与HBV引起的肝脏免疫损伤,但TIPE2在ACHBLF患者中的表达改变及意义目前尚无研究。研究目的 1.探讨ACHBLF患者外周血单个核细胞中TIPE2mRNA的表达改变,以及TIPE2与ACHBLF病情严重程度和疾病预后的关系； 2.初步探讨TIPE2影响ACHBLF病情发展的作用机制。研究方法本研究共纳入56例ACHBLF患者,60例CHB患者和24例健康对照者。我们对ACHBLF患者进行了3个月随访,根据患者3个月后的生存情况将患者分为存活组和死亡组。通过密度梯度离心法提取研究对象外周血单个核细胞,Trizol提取总RNA,采用RT-PCR法检测PBMCs中TIPE2mRNA的相对表达水平。另外,体外培养外周血单个核细胞,检测应用脂多糖(lipopolysaccharide, LPS)刺激后单个核细胞中TIPE2、IL-6和TNF-α mRNA的表达水平。促炎细胞因子IL-6和TNF-α是单个核细胞分泌的主要细胞因子,其体外分泌水平被认为可以代表机体细胞免疫水平。研究结果 1. ACHBLF患者PBMCs中TIPE2mRNA水平明显高于健康对照组(P0.05)和CHB患者(P0.01)。与健康对照相比,CHB患者TIPE2mRNA水平降低(P0.05)。 2. ACHBLF患者TIPE2mRNA表达水平与患者总胆红素水平(r=0.300,P=0.024)、凝血酶原时间的国际标准化比值(r=0.301,P=0.024)和终末期肝病模型积分(r=0.335,P=0.011)呈明显正相关关系,与谷丙转氨酶、白蛋白、肌酐水平、凝血酶原活动度、HBV-DNA病毒量和HBeAg均不具有相关性。 3. ACHBLF死亡组患者TIPE2表达明显高于存活组患者(P0.01)。用TIPE2预测ACHBLF患者预后,其受试者工作特征曲线下面积为0.721(95%CI0.572-0.870)。我们进一步研究了ACHBLF患者TIPE2表达的动态改变。收集32例ACHBLF患者入院第二天、第一周、第二周和第三周的连续血样标本,研究发现,18例存活患者入院三周内PBMCs TIPE2表达随病情好转降低,而14例死亡患者TIPE2表达不降反而升高。 4. TIPE2与ACHBLF患者细胞免疫功能下降有关。与健康对照和CHB患者相比,ACHBLF患者体外刺激的单个核细胞TIPE2mRNA表达水平明显增高(P均0.05),相反,IL-6和TNF-a mRNA的表达水平明显降低(P均0.05),在死亡组ACHBLF患者中这种趋势更为明显。另外,LPS刺激后外周血单个核细胞中TIPE2与TNF-α的表达呈负相关(r=-0.337,P=0.048)。结论： TIPE2在慢性HBV感染重症化过程中发挥重要作用。ACHBLF患者TIPE2基因表达水平与病情严重程度正相关,TIPE2基因表达增高是患者预后不良的预测指标。TIPE2可能通过负性调控细胞免疫反应介导ACHBLF的发生和发展,这为探寻新的ACHBLF治疗靶点提供了基础。
[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 - 纬 , 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 - 纬 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 - 伪 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 .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62;R575.3

【参考文献】