本文關(guān)鍵詞： NLRP3炎性體 單核巨噬細胞 肝功能衰竭 肝炎 固有免疫　出處：《河北醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:乙型肝炎病毒(hepatitis B virus,HBV)相關(guān)慢加急性肝功能衰竭(acute on chronic liver failure,ACLF)是在慢性乙型肝病基礎(chǔ)上出現(xiàn)的嚴重的肝細胞壞死、凋亡,繼而引起嚴重的肝功能損害和各種并發(fā)癥的臨床綜合征。其病情兇險,進展迅速,預后不良。目前大多數(shù)學者認為其發(fā)病機制與免疫損傷密切相關(guān)。有研究稱:在肝衰竭患者的外周血中存在“免疫紊亂”狀態(tài),但目前尚無確切的指標能夠明確患者的免疫狀態(tài)。核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體3(NACHT-LRR-PYD-containing proteins 3,NLRP3)炎性體是一種多蛋白復合體,屬于固有免疫系統(tǒng),通過識別外源性及內(nèi)源性損傷因子發(fā)揮作用。研究發(fā)現(xiàn)其在急性肝衰竭發(fā)揮促炎作用,但其在HBV相關(guān)ACLF中的作用尚未闡明。本研究通過檢測HBV相關(guān)ACLF患者外周血、肝組織中NLRP3、Caspase-1及細胞因子白細胞介素-1β(interleukin,IL-1β)、IL-18的表達,初步探討NLRP3炎性體在HBV相關(guān)ACLF患者中的作用。方法:選取2016年1月至2016年10月河北醫(yī)科大學第三醫(yī)院門診或住院患者,HBV相關(guān)ACLF患者30例(ACLF組)、CHB患者30例(CHB組)和健康體檢者15例(健康對照組)。此外,收集接受肝移植的HBV相關(guān)ACLF患者肝組織8例、肝組織活檢的CHB患者肝組織8例和正常供體肝組織5例。檢測入組患者血清ALB、ALT、AST、TB、DB、INR、HBsAg、HBeAg、HBV DNA水平。流式細胞學檢測外周血單個核細胞(peripheral blood mononuclear cell,PBMC)中NLRP3的表達量及表達位置。實時熒光定量(quantitative real-time)PCR檢測PBMC、肝組織中NLRP3、Caspase-1、IL-1β、IL-18mRNA的表達,并對PBMC進行磁珠分選,應用RT-qPCR分別檢測CD14+單核細胞、CD14-PBMC細胞群中NLRP3、Caspase-1、IL-1β、IL-18 mRNA的表達;免疫組織化學染色法檢測肝組織中NLRP3的表達以及與CD68共同表達。結(jié)果:1一般資料:各組研究對象年齡、性別比例具有可比性。ACLF組患者血清alt、ast、tb、db、inr水平明顯高于chb組及健康對照組(p0.01);aclf組患者alb、hbvdna水平明顯低于chb組(p0.01)。2乙型肝炎病毒相關(guān)慢加急性肝功能衰竭組外周血單個核細胞中nlrp3的表達下降應用流式細胞學技術(shù)分析各組患者外周血單核細胞中nlrp3的表達水平(mfi),結(jié)果顯示:aclf組nlrp3的表達量明顯低于chb組和健康對照組(p0.01)。chb組nlrp3的表達亦低于健康對照組(p0.01)。進一步分析aclf中晚期和aclf早期患者單核細胞中nlrp3的表達,結(jié)果顯示:aclf中晚期患者明顯低于aclf早期患者(p0.01)。應用rt-qpcr檢測pbmc中nlrp3的表達,結(jié)果顯示:aclf組nlrp3mrna水平低于chb組和健康對照組(p0.05)。chb組nlrp3mrna的表達亦低于健康對照組(p0.01)。3nlrp3主要表達在cd14+cd16+單核細胞群nlrp3在單核細胞亞群中的表達:在cd14++cd16-單核細胞群和cd16++cd14-單核細胞群,aclf組、chb組、健康對照組中nlrp3的表達未見明顯差異(p0.05);在cd14+cd16+單核細胞群,aclf組、chb組均低于健康對照組(p0.01),aclf組nlrp3的降低最為明顯,尤其是aclf中晚期患者。同時,應用cd14磁柱分離pbmc,分別檢測cd14+單核細胞、cd14-pbmc中nlrp3mrna水平。aclf組、chb組cd14+單核細胞中nlrp3mrna水平明顯低于健康對照組(p0.05),以aclf組降低顯著。而三組在cd14-pbmc細胞中nlrp3mrna的表達未見明顯差異(p0.05)。4乙型肝炎病毒相關(guān)慢加急性肝功能衰竭組肝組織中nlrp3的表達升高免疫組織化學染色結(jié)果顯示:aclf組肝組織中nlrp3的表達明顯高于chb組(p0.01),健康對照組肝組織未見nlrp3表達。nlrp3在匯管區(qū)與小葉內(nèi)均有表達,以小葉內(nèi)為主,cd68與nlrp3共染提示:nlrp3主要表達于肝小葉內(nèi)kupffer細胞胞漿。rt-qpcr檢測肝組織中nlrp3mrna的表達顯示:aclf組nlrp3mrna的表達高于chb組和健康對照組(p0.01)。chb組nlrp3mRNA的表達亦高于健康對照組(P0.01)。5乙型肝炎病毒相關(guān)慢加急性肝功能衰竭組外周血單個核細胞中Caspase-1及細胞因子IL-1β、IL-18的表達下降RT-qPCR檢測PBMC中Caspase-1及細胞因子IL-1β、IL-18的表達顯示:ACLF組Caspase-1、IL-1βmRNA水平均低于CHB組Caspase-1、IL-1β水平,IL-18 mRNA于兩組未見明顯差異(P0.05),ACLF組與健康對照組mRNA水平未見明顯差異(P0.05)。PT-q PCR檢測CD14磁珠分選后Caspase-1及細胞因子IL-1β、IL-18的表達顯示:ACLF組CD14+單核細胞Caspase-1、IL-1β、IL-18 mRNA的表達低于CHB組Caspase-1、IL-1β、IL-18 m RNA水平,ACLF組CD14-PBMC中Caspase-1、IL-1β、IL-18 mRNA的表達低于CHB組Caspase-1、IL-1β、IL-18 mRNA的表達。ACLF組與健康對照組未見明顯差異(P0.05)。6乙型肝炎病毒相關(guān)慢加急性肝功能衰竭組肝組織中Caspase-1及細胞因子IL-1β、IL-18的表達升高RT-qPCR檢測肝組織中Caspase-1及細胞因子IL-1β、IL-18mRNA的表達顯示:ACLF組IL-1βmRNA、IL-18mRNA的表達均高于CHB組及健康對照組(P0.05或P0.01)。ACLF組Caspase-1mRNA的表達與CHB組差異無統(tǒng)計學意義(P0.05)。7 ACLF組患者CD14+CD16+單核細胞NLRP3的表達量與患者MELD評分呈負相關(guān)(R=-0.74),相關(guān)具有統(tǒng)計學意義(P0.01)。結(jié)論:1 NLRP3炎性體在HBV相關(guān)慢加急性肝功能衰竭患者外周血表達降低,可能與免疫抑制有關(guān)。2 NLRP3主要表達于CD14+CD16+單核細胞,通過向肝臟募集,造成肝臟損傷。
[Abstract]:Objective: hepatitis B virus (hepatitis B, virus, HBV) related acute on chronic liver failure (acute on chronic liver failure, ACLF) is found in chronic hepatitis B on the basis of serious liver cell necrosis, apoptosis, clinical complications and cause severe liver damage and the syndrome. The disease risks rapid progression and poor prognosis. At present, most scholars believe that the pathogenesis and immune injury are closely related. The study said: "the immune disorder state in patients with liver failure in peripheral blood, but there is no exact index to the immune status of patients clearly. The nucleotide binding oligomerization domain (NACHT-LRR-PYD-containing like receptor 3 proteins 3, NLRP3) inflammasome is a multi protein complex, which belongs to the innate immune system play a role through the identification of exogenous and endogenous injury factor. Research found in acute Liver failure exert proinflammatory effects, but in the HBV the role of ACLF has not been elucidated. This study through the detection of HBV in peripheral blood of ACLF patients, NLRP3 in liver tissue, Caspase-1 and interleukin -1 beta (interleukin beta, IL-1), the expression of IL-18, a preliminary study of the NLRP3 inflammasome in HBV related ACLF patients. Methods: from January 2016 to October 2016 in the Third Hospital of Hebei Medical University outpatient or hospitalized patients, 30 cases of HBV ACLF patients (ACLF group), 30 cases of CHB patients (CHB group) and 15 healthy subjects (healthy control group). In addition, collected 8 cases of liver tissue of patients with HBV ACLF related liver transplantation, the liver tissue of patients with CHB liver biopsy in 8 patients and normal donor liver tissues in 5 cases. The patients detected serum ALB, ALT, AST, TB, DB, INR, HBsAg, HBeAg, HBV and DNA. The level of blood mononuclear cells by flow cytometry. The peripheral (peripheral blood mononuclea R cell, PBMC) and the position of the expression of NLRP3. Real time fluorescent quantitative detection of PBMC PCR (quantitative real-time), NLRP3, Caspase-1 in liver, IL-1 beta, IL-18mRNA expression, and sorting of PBMC were detected by RT-qPCR in CD14+ cells, NLRP3 CD14-PBMC cells, Caspase-1, IL-1 the expression of IL-18 beta, mRNA; immunohistochemistry method was used to detect the expression of NLRP3 in liver tissue and the expression of CD68. Results: 1 general information: each research object of age, sex ratio is comparable to that of.ACLF group patients serum ALT, AST, TB, DB, INR levels were significantly higher than those in CHB group and healthy controls group (P0.01); ACLF group of Alb patients, HBVDNA levels were significantly lower than group CHB (P0.01) NLRP3 expression of.2 of hepatitis B virus related acute on chronic liver failure group in peripheral blood mononuclear cells in patients with peripheral down were analyzed by flow cytometry technique The expression level of NLRP3 in blood mononuclear cells (MFI), the results showed that the expression of ACLF in group NLRP3 was significantly lower than that of CHB group and healthy control group (P0.01) group the expression of.Chb NLRP3 was lower than that of the healthy control group (P0.01). Further analysis of expression, and ACLF ACLF NLRP3 in the early stage of monocytes in patients the results showed that ACLF was significantly lower than that in patients with advanced ACLF patients (P0.01). The expression of NLRP3 was detected by RT-qPCR, PBMC results show that: the level of nlrp3mrna in ACLF group was lower than that of CHB group and healthy control group (P0.05) group the expression of.Chb nlrp3mrna was lower than that of healthy controls (P0.01).3nlrp3 is mainly expressed in cd14+cd16+ cells group NLRP3 on the expression of monocyte subsets in cd14++cd16- monocytes and mononuclear cells of cd16++cd14- group, ACLF group, CHB group, there was no significant difference between the expression of NLRP3 in healthy control group (P0.05); in mononuclear cells of cd14+cd16+ group, ACLF group, CHB Group were lower than the healthy control group (P0.01), NLRP3 of ACLF group decreased the most obvious, especially in patients with advanced ACLF. At the same time, the application of PBMC CD14 magnetic separation column, were detected in mononuclear cells of cd14+.Aclf group, nlrp3mrna level in cd14-pbmc CHB group, cd14+ nlrp3mrna in mononuclear cells was significantly lower than that of healthy controls (P0.05), in ACLF group decreased significantly. While the three group nlrp3mrna expression in cd14-pbmc cells showed no significant difference between the expression of NLRP3 (P0.05).4 of hepatitis B virus related acute on chronic liver failure group in liver tissue increased immunohistochemistry showed that the expression of NLRP3 in liver tissue in ACLF group was significantly higher than that of group CHB (P0.01), healthy control group liver tissue showed no NLRP3 expression of.Nlrp3 in the periportal and lobular expressed by small leaf, CD68 and NLRP3 were stained indicated that NLRP3 was mainly expressed in the liver small leaf in the cytoplasm of Kupffer cells.Rt-qpcr detection The expression of nlrp3mrna in liver tissue: the expression of ACLF in group nlrp3mrna was higher than that of CHB group and healthy control group (P0.01) group the expression of.Chb nlrp3mRNA was significantly higher than that in healthy control group (P0.01) beta Caspase-1 and cytokine IL-1.5 of hepatitis B virus related acute on chronic liver failure group in peripheral blood mononuclear cells, decreased IL-18 the expression of beta Caspase-1 and cytokine IL-1 RT-qPCR detection in PBMC showed that the expression of IL-18: group ACLF Caspase-1, IL-1 beta mRNA levels were lower than those in group CHB Caspase-1, IL-1 IL-18 mRNA beta level, in the two groups had no significant difference (P0.05), the control group mRNA levels showed no significant difference between ACLF group and health (P0.05).PT-q PCR detection of CD14 multisort after Caspase-1 and cytokines of IL-1 beta, showed that the expression of IL-18: ACLF group CD14+ monocytes Caspase-1, IL-1 beta, the expression of IL-18 mRNA was lower than that of CHB group Caspase-1, IL-1 IL-18 m beta, the level of RNA, ACLF in the CD14-PBMC group Caspase-1, IL-1 beta, IL-18 expression of mRNA was lower than that of CHB group Caspase-1, IL-1 IL-18 expression of mRNA beta,.ACLF group and the control group showed no significant difference (P0.05) beta Caspase-1 and cytokine IL-1.6 of hepatitis B virus related acute on chronic liver failure group in liver tissue, the expression of IL-18 and Caspase-1 increased beta cell factor detection of IL-1 RT-qPCR in liver tissues, the expression of IL-18mRNA showed: ACLF group IL-1 beta mRNA, IL-18mRNA expression was higher than that of CHB group and healthy control group (P0.05 or P0.01) had no significant difference between the expression of CHB and.ACLF group Caspase-1mRNA (P0.05) NLRP3 expression on monocytes in patients with MELD and.7 in ACLF group CD14+CD16+ the score was negatively correlated (R=-0.74), statistically significant correlation (P0.01). Conclusion: 1 patients with NLRP3 inflammasome in HBV related acute on chronic liver failure in peripheral blood decreased expression may be associated with immunosuppression.2 NLRP3 It is expressed in the CD14+CD16+ monocyte, which is raised by the liver to cause liver damage.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62;R575.3

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本文編號：1487234