NLRP3炎性体在HBV相关慢加急性肝功能衰竭中的作用

发布时间：2018-02-03 11:13

本文关键词： NLRP3炎性体单核巨噬细胞肝功能衰竭肝炎固有免疫　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)相关慢加急性肝功能衰竭(acute on chronic liver failure,ACLF)是在慢性乙型肝病基础上出现的严重的肝细胞坏死、凋亡,继而引起严重的肝功能损害和各种并发症的临床综合征。其病情凶险,进展迅速,预后不良。目前大多数学者认为其发病机制与免疫损伤密切相关。有研究称:在肝衰竭患者的外周血中存在“免疫紊乱”状态,但目前尚无确切的指标能够明确患者的免疫状态。核苷酸结合寡聚化结构域样受体3(NACHT-LRR-PYD-containing proteins 3,NLRP3)炎性体是一种多蛋白复合体,属于固有免疫系统,通过识别外源性及内源性损伤因子发挥作用。研究发现其在急性肝衰竭发挥促炎作用,但其在HBV相关ACLF中的作用尚未阐明。本研究通过检测HBV相关ACLF患者外周血、肝组织中NLRP3、Caspase-1及细胞因子白细胞介素-1β(interleukin,IL-1β)、IL-18的表达,初步探讨NLRP3炎性体在HBV相关ACLF患者中的作用。方法:选取2016年1月至2016年10月河北医科大学第三医院门诊或住院患者,HBV相关ACLF患者30例(ACLF组)、CHB患者30例(CHB组)和健康体检者15例(健康对照组)。此外,收集接受肝移植的HBV相关ACLF患者肝组织8例、肝组织活检的CHB患者肝组织8例和正常供体肝组织5例。检测入组患者血清ALB、ALT、AST、TB、DB、INR、HBsAg、HBeAg、HBV DNA水平。流式细胞学检测外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中NLRP3的表达量及表达位置。实时荧光定量(quantitative real-time)PCR检测PBMC、肝组织中NLRP3、Caspase-1、IL-1β、IL-18mRNA的表达,并对PBMC进行磁珠分选,应用RT-qPCR分别检测CD14+单核细胞、CD14-PBMC细胞群中NLRP3、Caspase-1、IL-1β、IL-18 mRNA的表达;免疫组织化学染色法检测肝组织中NLRP3的表达以及与CD68共同表达。结果:1一般资料:各组研究对象年龄、性别比例具有可比性。ACLF组患者血清alt、ast、tb、db、inr水平明显高于chb组及健康对照组(p0.01);aclf组患者alb、hbvdna水平明显低于chb组(p0.01)。2乙型肝炎病毒相关慢加急性肝功能衰竭组外周血单个核细胞中nlrp3的表达下降应用流式细胞学技术分析各组患者外周血单核细胞中nlrp3的表达水平(mfi),结果显示:aclf组nlrp3的表达量明显低于chb组和健康对照组(p0.01)。chb组nlrp3的表达亦低于健康对照组(p0.01)。进一步分析aclf中晚期和aclf早期患者单核细胞中nlrp3的表达,结果显示:aclf中晚期患者明显低于aclf早期患者(p0.01)。应用rt-qpcr检测pbmc中nlrp3的表达,结果显示:aclf组nlrp3mrna水平低于chb组和健康对照组(p0.05)。chb组nlrp3mrna的表达亦低于健康对照组(p0.01)。3nlrp3主要表达在cd14+cd16+单核细胞群nlrp3在单核细胞亚群中的表达:在cd14++cd16-单核细胞群和cd16++cd14-单核细胞群,aclf组、chb组、健康对照组中nlrp3的表达未见明显差异(p0.05);在cd14+cd16+单核细胞群,aclf组、chb组均低于健康对照组(p0.01),aclf组nlrp3的降低最为明显,尤其是aclf中晚期患者。同时,应用cd14磁柱分离pbmc,分别检测cd14+单核细胞、cd14-pbmc中nlrp3mrna水平。aclf组、chb组cd14+单核细胞中nlrp3mrna水平明显低于健康对照组(p0.05),以aclf组降低显著。而三组在cd14-pbmc细胞中nlrp3mrna的表达未见明显差异(p0.05)。4乙型肝炎病毒相关慢加急性肝功能衰竭组肝组织中nlrp3的表达升高免疫组织化学染色结果显示:aclf组肝组织中nlrp3的表达明显高于chb组(p0.01),健康对照组肝组织未见nlrp3表达。nlrp3在汇管区与小叶内均有表达,以小叶内为主,cd68与nlrp3共染提示:nlrp3主要表达于肝小叶内kupffer细胞胞浆。rt-qpcr检测肝组织中nlrp3mrna的表达显示:aclf组nlrp3mrna的表达高于chb组和健康对照组(p0.01)。chb组nlrp3mRNA的表达亦高于健康对照组(P0.01)。5乙型肝炎病毒相关慢加急性肝功能衰竭组外周血单个核细胞中Caspase-1及细胞因子IL-1β、IL-18的表达下降RT-qPCR检测PBMC中Caspase-1及细胞因子IL-1β、IL-18的表达显示:ACLF组Caspase-1、IL-1βmRNA水平均低于CHB组Caspase-1、IL-1β水平,IL-18 mRNA于两组未见明显差异(P0.05),ACLF组与健康对照组mRNA水平未见明显差异(P0.05)。PT-q PCR检测CD14磁珠分选后Caspase-1及细胞因子IL-1β、IL-18的表达显示:ACLF组CD14+单核细胞Caspase-1、IL-1β、IL-18 mRNA的表达低于CHB组Caspase-1、IL-1β、IL-18 m RNA水平,ACLF组CD14-PBMC中Caspase-1、IL-1β、IL-18 mRNA的表达低于CHB组Caspase-1、IL-1β、IL-18 mRNA的表达。ACLF组与健康对照组未见明显差异(P0.05)。6乙型肝炎病毒相关慢加急性肝功能衰竭组肝组织中Caspase-1及细胞因子IL-1β、IL-18的表达升高RT-qPCR检测肝组织中Caspase-1及细胞因子IL-1β、IL-18mRNA的表达显示:ACLF组IL-1βmRNA、IL-18mRNA的表达均高于CHB组及健康对照组(P0.05或P0.01)。ACLF组Caspase-1mRNA的表达与CHB组差异无统计学意义(P0.05)。7 ACLF组患者CD14+CD16+单核细胞NLRP3的表达量与患者MELD评分呈负相关(R=-0.74),相关具有统计学意义(P0.01)。结论:1 NLRP3炎性体在HBV相关慢加急性肝功能衰竭患者外周血表达降低,可能与免疫抑制有关。2 NLRP3主要表达于CD14+CD16+单核细胞,通过向肝脏募集,造成肝脏损伤。
[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.

【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;R575.3

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