本文關(guān)鍵詞： 黑色素瘤缺乏因子-2 炎癥小體 肝炎乙型慢性 肝功能衰竭 固有免疫 炎癥損傷　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:乙型肝炎病毒(hepatitis B virus,HBV)是人類嗜肝病毒屬中重要的一員,慢性HBV感染可以導(dǎo)致慢性乙型病毒性肝炎(chronic hepatitis B,CHB),肝硬化和肝細(xì)胞肝癌(hepatocellular carcinoma,HCC)。導(dǎo)致肝臟損傷的原因不是HBV復(fù)制直接破壞肝細(xì)胞。目前大多數(shù)的觀點(diǎn)認(rèn)為機(jī)體免疫調(diào)控的紊亂是導(dǎo)致慢性肝損傷的主要原因,然而對固有免疫在慢乙肝肝臟損傷中的作用的認(rèn)識尚不清楚。胞質(zhì)雙鏈DNA感應(yīng)蛋白黑色素瘤缺乏因子-2(absent in melanoma-2,AIM2)是一種主要定位于細(xì)胞質(zhì)的蛋白質(zhì)。AIM2可與胞質(zhì)內(nèi)雙鏈DNA(double-stranded DNA,dsDNA)結(jié)合,并且連接適配器分子凋亡相關(guān)斑點(diǎn)樣蛋白(apoptosis-associated speck-like protein containing a CARD,ASC),形成炎癥小體,激活半胱氨酸天冬氨酸蛋白酶-1(Caspase-1,CASP-1),繼而引起成熟的白細(xì)胞介素1β(interleukin 1β,IL-1β)和白細(xì)胞介素18(interleukin,IL-18)產(chǎn)生。本研究主要通過觀察AIM2是否在慢性乙型肝炎患者肝臟組織中表達(dá),并且檢測AIM2 mRNA在肝組織和外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMCs)中的水平變化,初步探討HBV是否可通過激活A(yù)IM2-ASC-Caspase-1途徑,釋放炎性因子,導(dǎo)致肝細(xì)胞損傷,從固有免疫角度分析慢性乙型病毒性肝炎肝臟炎癥損傷的發(fā)病機(jī)制。方法:自2015年6月至2016年12月從河北醫(yī)科大學(xué)第三醫(yī)院感染科門診及住院患者隨機(jī)選取CHB患者30例,HBV相關(guān)慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)患者23例,征集同期于本醫(yī)院健康體檢者20例作為健康對照,收集所有觀察對象晨起空腹外周血血清。同時(shí)收集接受肝組織穿刺術(shù)的CHB患者肝組織標(biāo)本21例,接受肝移植術(shù)的HBV相關(guān)ACLF患者肝組織標(biāo)本19例,以及健康供肝肝組織5例。所納入患者入組前6個(gè)月均未曾接受免疫調(diào)節(jié)、抗病毒及乙肝疫苗治療。CHB診斷符合中華醫(yī)學(xué)會肝病學(xué)分會、中華醫(yī)學(xué)會感染病學(xué)分會制定的《慢性乙型肝炎防治指南(2015版)》,HBV相關(guān)ACLF診斷符合中華醫(yī)學(xué)會感染病學(xué)分會肝衰竭與人工肝學(xué)組、中華醫(yī)學(xué)會肝病學(xué)分會重型肝病與人工肝學(xué)組共同制定的《肝衰竭診療指南(2012年版)》。收集各組研究對象常規(guī)生化學(xué)指標(biāo),如白蛋白(albumin,alb)、丙氨酸氨基轉(zhuǎn)移酶(alaninetransaminase,alt)、天門冬氨酸氨基轉(zhuǎn)移酶(aspartatetransaminase,ast)、總膽紅素(totalbilirubin,tb)、直接膽紅素(directbilirubin,db)等,檢測chb患者及hbv相關(guān)aclf患者血清hbvdna載量、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。應(yīng)用免疫組織化學(xué)方法檢測chb患者及hbv相關(guān)aclf患者肝組織中aim2表達(dá),并采用實(shí)時(shí)熒光定量pcr檢測肝組織和外周血單個(gè)核細(xì)胞中aim2mrna表達(dá)水平。結(jié)果:1各組研究對象的人口學(xué)及臨床資料各組研究對象年齡及性別比例均具有可比性。chb組患者血清alb、hbvdna、hbsag、hbeag水平明顯高于hbv相關(guān)aclf組(p0.05),tb、db、inr水平均顯著低于hbv相關(guān)aclf組(p0.01),chb組與hbv相關(guān)aclf組間血清alt、ast水平無明顯差異(p0.05)。各組人口學(xué)指標(biāo)及臨床資料詳見table1。2慢性乙型病毒性肝炎組患者pbmc中aim2表達(dá)增加應(yīng)用實(shí)時(shí)熒光定量pcr檢測73名研究對象pbmc內(nèi)aim2mrna水平。aim2mrna在正常對照組相對表達(dá)量設(shè)定為1.00。chb組患者pbmc內(nèi)aim2mrna水平(4.426±5.395)明顯高于hbv相關(guān)aclf組(2.077±1.291)及健康對照組(1.00±0.00)(p均0.01)。hbv相關(guān)aclf組患者pbmc內(nèi)aim2mrna水平明顯高于健康對照組(p0.01)。各組pbmc內(nèi)aim2mrna表達(dá)水平詳見fig.1。3慢性乙型病毒性肝炎組患者肝組織中aim2表達(dá)增加應(yīng)用實(shí)時(shí)熒光定量pcr檢測45名研究對象肝組織內(nèi)aim2mrna水平。aim2mrna在正常對照組相對表達(dá)量設(shè)定為1.00。chb組患者肝組織內(nèi)aim2mrna水平(3.231±2.005)明顯高于hbv相關(guān)aclf組(1.598±0.662)及健康對照組(1.00±0.00)(p均0.01)。hbv相關(guān)aclf組患者肝組織內(nèi)aim2mrna水平明顯高于健康對照組(p0.01)。各組肝組織內(nèi)aim2mrna表達(dá)水平詳見fig.2。應(yīng)用免疫組織化學(xué)法檢測肝組織中AIM2的表達(dá)和分布。AIM2陽性表達(dá)主要分布在肝細(xì)胞胞質(zhì)內(nèi),顯微鏡下呈棕黃色顆粒狀。AIM2在正常肝組織幾乎不表達(dá),CHB組患者肝組織中AIM2表達(dá)較HBV相關(guān)ACLF組顯著增加。各組肝組織內(nèi)AIM2表達(dá)和分布詳見Fig.3(A)-(C)。4慢性乙型病毒性肝炎組患者PBMC中AIM2 mRNA表達(dá)水平與血清ALT、AST、HBV DNA、HBsAg、HBeAg的相關(guān)性分析CHB組患者PBMC AIM2 mRNA水平與血清ALT、AST水平均呈正相關(guān)(r=0.575,0.563,P均0.01)。CHB組患者PBMC AIM2 mRNA水平與血清HBV DNA、HBsAg、HBeAg水平均呈負(fù)相關(guān)(r=-0.663,-0.622,-0.431,P均0.01)。CHB組患者PBMC AIM2 mRNA水平與臨床指標(biāo)相關(guān)性分析詳見Fig.4(A)-(E)。結(jié)論:1慢性乙型病毒性肝炎患者肝組織和PBMC中AIM2均較HBV相關(guān)慢加急性肝衰竭患者表達(dá)增多,可能與慢乙肝較強(qiáng)的AIM2相關(guān)固有免疫應(yīng)答有關(guān)。2肝細(xì)胞胞質(zhì)中HBV DNA可能通過激活A(yù)IM2的表達(dá),促進(jìn)炎癥小體形成,誘導(dǎo)固有免疫反應(yīng),推測AIM2可能在慢性乙型病毒性肝炎肝臟炎癥損傷機(jī)制中發(fā)揮重要作用。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.62

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