慢性乙型肝炎患者黑色素瘤缺乏因子-2水平变化及其临床意义

发布时间：2018-01-25 04:26

本文关键词： 黑色素瘤缺乏因子-2 炎症小体肝炎乙型慢性肝功能衰竭固有免疫炎症损伤　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:乙型肝炎病毒(hepatitis B virus,HBV)是人类嗜肝病毒属中重要的一员,慢性HBV感染可以导致慢性乙型病毒性肝炎(chronic hepatitis B,CHB),肝硬化和肝细胞肝癌(hepatocellular carcinoma,HCC)。导致肝脏损伤的原因不是HBV复制直接破坏肝细胞。目前大多数的观点认为机体免疫调控的紊乱是导致慢性肝损伤的主要原因,然而对固有免疫在慢乙肝肝脏损伤中的作用的认识尚不清楚。胞质双链DNA感应蛋白黑色素瘤缺乏因子-2(absent in melanoma-2,AIM2)是一种主要定位于细胞质的蛋白质。AIM2可与胞质内双链DNA(double-stranded DNA,dsDNA)结合,并且连接适配器分子凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC),形成炎症小体,激活半胱氨酸天冬氨酸蛋白酶-1(Caspase-1,CASP-1),继而引起成熟的白细胞介素1β(interleukin 1β,IL-1β)和白细胞介素18(interleukin,IL-18)产生。本研究主要通过观察AIM2是否在慢性乙型肝炎患者肝脏组织中表达,并且检测AIM2 mRNA在肝组织和外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中的水平变化,初步探讨HBV是否可通过激活AIM2-ASC-Caspase-1途径,释放炎性因子,导致肝细胞损伤,从固有免疫角度分析慢性乙型病毒性肝炎肝脏炎症损伤的发病机制。方法:自2015年6月至2016年12月从河北医科大学第三医院感染科门诊及住院患者随机选取CHB患者30例,HBV相关慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)患者23例,征集同期于本医院健康体检者20例作为健康对照,收集所有观察对象晨起空腹外周血血清。同时收集接受肝组织穿刺术的CHB患者肝组织标本21例,接受肝移植术的HBV相关ACLF患者肝组织标本19例,以及健康供肝肝组织5例。所纳入患者入组前6个月均未曾接受免疫调节、抗病毒及乙肝疫苗治疗。CHB诊断符合中华医学会肝病学分会、中华医学会感染病学分会制定的《慢性乙型肝炎防治指南(2015版)》,HBV相关ACLF诊断符合中华医学会感染病学分会肝衰竭与人工肝学组、中华医学会肝病学分会重型肝病与人工肝学组共同制定的《肝衰竭诊疗指南(2012年版)》。收集各组研究对象常规生化学指标,如白蛋白(albumin,alb)、丙氨酸氨基转移酶(alaninetransaminase,alt)、天门冬氨酸氨基转移酶(aspartatetransaminase,ast)、总胆红素(totalbilirubin,tb)、直接胆红素(directbilirubin,db)等,检测chb患者及hbv相关aclf患者血清hbvdna载量、乙型肝炎病毒表面抗原(hepatitisbvirussurfaceantigen,hbsag)及乙型肝炎病毒e抗原(hepatitisbviruseantigen,hbeag)水平。应用免疫组织化学方法检测chb患者及hbv相关aclf患者肝组织中aim2表达,并采用实时荧光定量pcr检测肝组织和外周血单个核细胞中aim2mrna表达水平。结果:1各组研究对象的人口学及临床资料各组研究对象年龄及性别比例均具有可比性。chb组患者血清alb、hbvdna、hbsag、hbeag水平明显高于hbv相关aclf组(p0.05),tb、db、inr水平均显著低于hbv相关aclf组(p0.01),chb组与hbv相关aclf组间血清alt、ast水平无明显差异(p0.05)。各组人口学指标及临床资料详见table1。2慢性乙型病毒性肝炎组患者pbmc中aim2表达增加应用实时荧光定量pcr检测73名研究对象pbmc内aim2mrna水平。aim2mrna在正常对照组相对表达量设定为1.00。chb组患者pbmc内aim2mrna水平(4.426±5.395)明显高于hbv相关aclf组(2.077±1.291)及健康对照组(1.00±0.00)(p均0.01)。hbv相关aclf组患者pbmc内aim2mrna水平明显高于健康对照组(p0.01)。各组pbmc内aim2mrna表达水平详见fig.1。3慢性乙型病毒性肝炎组患者肝组织中aim2表达增加应用实时荧光定量pcr检测45名研究对象肝组织内aim2mrna水平。aim2mrna在正常对照组相对表达量设定为1.00。chb组患者肝组织内aim2mrna水平(3.231±2.005)明显高于hbv相关aclf组(1.598±0.662)及健康对照组(1.00±0.00)(p均0.01)。hbv相关aclf组患者肝组织内aim2mrna水平明显高于健康对照组(p0.01)。各组肝组织内aim2mrna表达水平详见fig.2。应用免疫组织化学法检测肝组织中AIM2的表达和分布。AIM2阳性表达主要分布在肝细胞胞质内,显微镜下呈棕黄色颗粒状。AIM2在正常肝组织几乎不表达,CHB组患者肝组织中AIM2表达较HBV相关ACLF组显著增加。各组肝组织内AIM2表达和分布详见Fig.3(A)-(C)。4慢性乙型病毒性肝炎组患者PBMC中AIM2 mRNA表达水平与血清ALT、AST、HBV DNA、HBsAg、HBeAg的相关性分析CHB组患者PBMC AIM2 mRNA水平与血清ALT、AST水平均呈正相关(r=0.575,0.563,P均0.01)。CHB组患者PBMC AIM2 mRNA水平与血清HBV DNA、HBsAg、HBeAg水平均呈负相关(r=-0.663,-0.622,-0.431,P均0.01)。CHB组患者PBMC AIM2 mRNA水平与临床指标相关性分析详见Fig.4(A)-(E)。结论:1慢性乙型病毒性肝炎患者肝组织和PBMC中AIM2均较HBV相关慢加急性肝衰竭患者表达增多,可能与慢乙肝较强的AIM2相关固有免疫应答有关。2肝细胞胞质中HBV DNA可能通过激活AIM2的表达,促进炎症小体形成,诱导固有免疫反应,推测AIM2可能在慢性乙型病毒性肝炎肝脏炎症损伤机制中发挥重要作用。
[Abstract]:Objective: hepatitis B virus (hepatitis B virus, HBV) is an important part of human eosinophil Hepatovirus, chronic HBV infection can lead to chronic hepatitis B (chronic hepatitis, B, CHB), liver cirrhosis and hepatocellular carcinoma (hepatocellular carcinoma, HCC). The cause of liver injury is not the direct destruction of HBV replication liver cells. Most of the view that the immune regulation disorder is a major cause of chronic liver injury, but the understanding of the role of innate immune in liver injury in chronic hepatitis B is not clear. The cytoplasmic double stranded DNA protein induced melanoma factor -2 (absent in lack melanoma-2, AIM2) is one of the main the.AIM2 protein located in the cytoplasm and cytoplasmic double stranded DNA (double-stranded DNA dsDNA) together, and connect the adapter molecules apoptosis associated specklike protein (apoptosis-associated speck-like prote In containing a CARD, ASC), the formation of inflammatory corpuscle, activation of caspase -1 (Caspase-1, CASP-1), followed by interleukin 1 beta (mature interleukin 1 beta, beta IL-1) and interleukin 18 (interleukin, IL-18). This study mainly by observing whether AIM2 the expression in liver tissue of patients with chronic hepatitis B, and detection of AIM2 mRNA in liver tissue and peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) levels, to explore the possibility of HBV could activate the AIM2-ASC-Caspase-1 pathway, the release of inflammatory factors that cause liver cell injury and analysis of the pathogenesis of chronic hepatitis B virus hepatitis B liver inflammation injury from the perspective of innate immunity. Methods: Department of infectious diseases from the Third Hospital of Hebei Medical University from June 2015 to December 2016 and were randomly selected 30 patients with CHB, HBV slow Urgent liver failure (acute-on-chronic liver failure, ACLF) in 23 cases, for the same period in our hospital 20 cases of healthy people as healthy control, collect all the observed objects of fasting peripheral blood serum. At the same time received liver tissue puncture of liver tissue of patients with CHB were 21 cases, 19 cases underwent liver transplantation HBV ACLF in liver tissue from patients and healthy donor liver tissues in 5 cases. The patients in group 6 months before had never received immune regulation, antiviral treatment of hepatitis B vaccine and.CHB diagnosis of Hepatology of Chinese Medical Association, Chinese Medical Association Branch of infectious diseases develop "chronic hepatitis B (2015 Edition) >, HBV ACLF diagnosis of infectious diseasese liver failure and artificial liver group, Chinese Medical Association, Chinese Medical Association guidelines for diagnosis and treatment of liver failure Hepatology of severe liver diseases and artificial liver group jointly developed" (2 The 012 Year Edition). The research object was collected conventional biochemical indicators, such as albumin (albumin, ALB), alanine aminotransferase (alaninetransaminase, ALT), aspartate aminotransferase (aspartatetransaminase, AST), total bilirubin (totalbilirubin, TB), direct bilirubin (directbilirubin, DB), detection of CHB patients HBV and ACLF in serum of patients with HBVDNA load, hepatitis B virus surface antigen (hepatitisbvirussurfaceantigen, HBsAg) and hepatitis B virus e antigen (hepatitisbviruseantigen, HBeAg). The expression level of aim2 was detected by immunohistochemical method in CHB patients and HBV in liver tissue of ACLF patients, and the level of expression of aim2mrna in liver tissue were detected by real-time fluorescence quantitative PCR and in the peripheral blood mononuclear cells. Results: 1 demographic and clinical data of each research object was the research object of age and sex ratio were comparable. CHB group of serum ALB, HBVDNA, HBsAg, HBeAg levels were significantly higher than that of HBV group ACLF (P0.05), TB, DB, INR were significantly lower than HBV group ACLF (P0.01), CHB group and HBV group ACLF serum ALT level of AST showed no significant difference (P0.05). The increased expression of aim2 by real time fluorescence quantitative PCR detection of 73 subjects in the PBMC level of aim2mrna.Aim2mrna in the normal control group the relative expression quantity is set to the aim2mrna level of 1.00.chb group in PBMC group and table1.2 clinical data for demographic indicators of chronic hepatitis B patients in group PBMC (4.426 + 5.395) was significantly higher than that of HBV group ACLF (2.077 + 1.291) and health the control group (1 + 0) (P 0.01) level in patients with PBMC.Hbv aim2mrna group ACLF was significantly higher than the control group (P0.01). The expression level of aim2 aim2mrna fig.1.3 in chronic hepatitis B patients in liver tissue were PBMC The increased expression of real-time PCR was used to detect the level of aim2mrna.Aim2mrna in liver tissue of 45 subjects in the normal control group the relative expression level of 1.00.chb group were set to aim2mrna in the liver tissue (3.231 + 2.005) was significantly higher than that of HBV group ACLF (1.598 + 0.662) and healthy controls (1 + 0) (P < 0.01 the level of aim2mrna).Hbv related ACLF patients in liver tissue were significantly higher than the control group (P0.01). The liver tissue expression of aim2mrna in the expression and distribution of.AIM2 AIM2 positive expression level of fig.2. in liver tissue was detected by immunohistochemistry in the liver is mainly distributed in the cytoplasm, brownish yellow granular.AIM2 almost no expression in normal the liver tissue was under the microscope, compared with HBV group ACLF significantly increased the expression of AIM2 in liver tissues of patients with CHB group. The expression of AIM2 in liver tissue in each group and distribution in Fig.3 (A) - (C).4 viral chronic hepatitis liver The level of serum ALT, AIM2 mRNA expression of inflammation in patients with PBMC AST, HBV DNA, HBsAg, HBeAg correlation analysis of CHB patients in the PBMC group AIM2 mRNA levels were positively correlated with serum ALT, AST levels (r=0.575,0.563, P 0.01).CHB AIM2 mRNA PBMC in patients with serum HBV level of DNA, HBsAg, negative correlation the level of HBeAg (r=-0.663, -0.622, -0.431 were P, 0.01) in.CHB group were PBMC AIM2 correlation between mRNA level and clinical index analysis. Fig.4 (A) - (E). Conclusion: 1 liver tissues of patients with chronic hepatitis B and PBMC in AIM2 were related to HBV in patients with acute on chronic liver failure was increased. May HBV DNA AIM2 related chronic hepatitis B with strong innate immune response on.2 liver cells in the cytoplasm may be through the activation of AIM2 expression, promote inflammasome formation, induce innate immune responses, suggesting that AIM2 may in the mechanism of chronic viral hepatitis in liver inflammatory injury Play an important role.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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