黑色素瘤缺乏因子2在慢性乙型肝炎及其相关性肾小球肾炎中作用机制的初步研究

发布时间：2018-03-08 12:12

  本文选题：慢性乙型肝炎　切入点：HBV相关性肾小球肾炎　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：[背景/目的]HBV感染是个全球性问题,由于HBV感染所导致的肝硬化,肝衰竭甚至肝癌等临床结局严重的威胁人类的健康。同时,HBV的感染也可导致许多肝外疾病的发生,HBV相关性肾小球肾炎即是常见的一种肝外HBV感染相关疾病。目前,关于两种疾病的发病机制均不明确,大多数观点认为与免疫反应相关。黑色素缺乏因子2 (AIM2)属于HIN-200蛋白家族,其主要存在于细胞浆中,AIM2可被胞浆内dsDNA所识别并激活,并结合中间蛋白ASC形成炎性复合体,进一步激活caspase-1途径,诱导IL-1β、IL-18等炎性因子的成熟和释放,最终导致炎症的发生,在机体的固有免疫反应而诱发的炎症中扮演重要作用。在慢性乙型肝炎中,乙肝病毒在复制的过程,首先位于肝细胞核内的CCC-DNA为乙肝病毒复制的终模板,由其为模板合成负链DNA,再由负链DNA为模板合成正链DNA,然后形成ds-DNA,在由胞内向胞外转移的过程中,利用胞内蛋白完成病毒的装配而释放。那么,在转运的过程中ds-DNA便有机会被胞浆内的AIM2所识别,形成炎性复合体从而诱发肝脏炎症;同样,在HBV-GN中,既往已有研究报道电镜检测可以发现肾组织固有细胞内存在有乙肝病毒样颗粒及HBV-DNA的复制,那么肾脏固有细胞胞浆中的AIM2也有机会识别HBV-DNA并被激活,通过caspase-1途径,释放IL-1β、IL-18炎性因子,从而导致肾脏炎症的发生。为了验证这一设想,我们进行了此项研究,通过检测肝脏及肾脏内AIM2的表达及与caspase-1,IL-1 β及IL-18表达的关系,探讨HBV与AIM2的激活在CHB、HBV-GN中可能的致病机制,为这一疾病的发病机制和治疗提供新的思路和研究方法。[方法]研究通过组织免疫组化实验测定慢性乙型肝炎组织及HBV-GN的肾组织中AIM2水平,及其与caspase-1,IL-1 β、IL-18等炎性因子表达水平以及肝细胞,肾脏炎症程度的关系。共计70例慢性肝炎的患者及79例慢性肾小球肾炎的患者纳入到本项研究,其中在慢性肝炎患者中,慢性乙型肝炎47例为实验组,慢性丙肝患者23例为对照组;慢性肾小球肾炎的患者中,慢性乙肝相关性肾炎(HBV-GN)病人54例作为实验组,慢性肾小球肾炎(CGN)患者25例作为对照组。以上所有的病人均根据研究的要求行肝脏穿刺或肾脏穿刺取得肝或肾组织标本制备石蜡包埋切片。应用免疫组化法半定量测定患者肝、肾穿标本中AIM2、Caspase-1、IL-18、IL-1p的表达,同时分析AIM2的表达水平与临床资料的相关性。本研究结果运用SPSS 17.0软件包进行数据的统计学处理,两组间AIM2的表达及临床资料的的关系采用χ2检验和Fisher精确概率检验,多个样本间的单因素比较采用单因素方差分析,相关性分析采用Spearman's检验。所有结果的分析均以P0.05为有统计学意义。[结果]1、在慢性乙型肝炎组织中,AIM2的表达主要定位于肝细胞胞浆内;2、AIM2在慢乙肝组织内的表达明显高于其在慢丙肝患者肝组织中的表达(89.4%vs. 8.7%,p0.00),两者具有统计学意义;3、慢乙肝患者肝组织内AIM2的表达与年龄(p=0.178)、性别(p=0.148)、e抗原的状态(p=0.825)无关;4、AIM2的表达与ALT的水平具有统计学意义(p=0.03),而与AST的水平无关(p=0.378);5、在HBV高载量组(HBV-DNA≥1×105copies/ml), AIM2的表达高于其在HBV低载量组(HBV-DNA1×105 copies/ml)的表达(p0.01),两者具有统计学差异;6、AIM2的表达与肝脏炎症分级存在高度的相关性(p=0.007),而和纤维化分级则无明显相关(p=0.101);7、慢乙肝肝组织内 AIM2 的表达与 caspase-1(rs=0.738,p0.01),IL-1β(rs=0.527,p0.01)及 IL-18 (rs=0.642,p0.01)的表达呈正相关;8、AIM2在HBV-GN肾组织中主要定位于肾小球系膜细胞及内皮细胞内;9、在HBV-GN中,AIM2的阳性表达率为81. 4%,明显高于其在CGN中的表达(4.0%),两者具有统计学差异(81.4% vs. 4.0%, p0. 01);10、AIM2的表达与血清e抗原的状态无关(p=0. 614);同时与肾组织中HBV相关抗原的沉积无关(p=0. 511);11、HBV-GN中高病毒载量组中AIM2的表达相比较于低病毒载量组有统计学差异(p0. 05);12、不同病理类型的HBV-GN中AIM2的表达无统计学差异(p=0. 940);13、HBV-GN肾组织中AIM2的表达分别与caspase-1的表达(rs = 0.444, p 0.01)以及IL-1β的表达(rs=0.379,p0.01)呈正相关;而caspase-1的表达亦与IL-]β的表达呈正相关(rs = 0.515, p 0.01)。[结论]AIM2可能通过识别并结合HBV-DNA激活后活化Caspase-]形成炎性复合体,释放致前炎性因子IL-1 β,IL-18造成CHB肝脏损伤及HBV-GN的肾损伤。
[Abstract]:Background / purpose of]HBV infection is a global problem due to HBV infection caused by liver cirrhosis, liver failure and liver cancer clinical outcomes such as a serious threat to human health. At the same time, HBV infection may cause many extrahepatic disease, HBV associated glomerulonephritis is a common extrahepatic infection related HBV the pathogenesis of disease. At present, about two kinds of diseases are not clear, the majority view is related to immune response. The lack of melanin factor 2 (AIM2) belongs to HIN-200 protein family, which mainly exists in the cytoplasm, AIM2 was in the cytoplasm of dsDNA recognition and activation, and the formation of inflammatory protein complex combined with intermediate ASC, further activation of the caspase-1 pathway, induced by beta IL-1, IL-18 and other inflammatory factors resulting in the maturation and release of inflammation in the natural immune response induced inflammation plays an important role in chronic B. Hepatitis B, hepatitis B virus replication in the process, final template first located in the nuclei of hepatocytes CCC-DNA for hepatitis B virus replication, the template for synthesis of negative strand DNA, and then by the negative strand DNA as template to synthesize positive strand DNA, and then the formation of ds-DNA, the transfer from the cell to the extracellular medium, the completion of the assembly the virus using intracellular protein and release. Then, in the transport process of ds-DNA will have a chance to be in the cytoplasm of the AIM2 recognition, the formation of inflammatory complexes to induce inflammation of the liver; similarly, in HBV-GN, the previous research reports of SEM detection found hepatitis B virus like particles and replication of HBV-DNA in renal tissue inherent in the cell, then the renal cells in the cytoplasm of AIM2 also have the opportunity to identify HBV-DNA and activated by the caspase-1 pathway, the release of IL-1 beta IL-18, inflammatory cytokines, leading to kidney inflammation. In order to verify this idea, We conducted this study, by detecting the expression of liver and kidney in AIM2 and caspase-1, IL-1 beta and IL-18 expression, to investigate the HBV and AIM2 activation in CHB, the possible mechanism of HBV-GN, the level of AIM2 provides new ideas and research methods. The research method of renal tissue determination of tissue of chronic hepatitis hepatitis and HBV-GN by immunohistochemical experiments in pathogenesis and treatment of this disease, and caspase-1, IL-1 beta, IL-18 expression levels of inflammatory cytokines and liver cells, inflammation degree of kidney. A total of 70 cases of chronic hepatitis B patients and 79 cases of chronic glomerulonephritis patients into the study in patients with chronic hepatitis, 47 cases of chronic hepatitis B chronic hepatitis C patients as the experimental group, 23 patients in the control group; chronic glomerulonephritis in patients with chronic hepatitis B virus associated glomerulonephritis (HBV-GN) patients 54 cases as The experimental group, chronic glomerulonephritis (CGN) patients 25 cases as control group. All patients were based on the requirements for liver puncture or kidney puncture to obtain liver or kidney tissue preparation paraffin. Immunohistochemical method semi quantitative determination in patients with liver and kidney in the AIM2, Caspase-1. The expression of IL-1p, IL-18, correlation analysis and the expression of AIM2 and clinical data. The results of this study using the SPSS 17 software package was used for statistical processing of data, the relationship between the clinical data and the expression of AIM2 between the two groups using the 2 test and Fisher exact test, multiple samples between the single factor analysis was used to compare one way ANOVA, correlation analysis using Spearman's test. All the results were analyzed by P0.05 statistical significance. Results]1 in chronic hepatitis B, the expression of AIM2 was mainly located in the cytoplasm of liver cells; 2, the expression of AIM2 in chronic hepatitis tissues was significantly higher than the expression in liver tissue of patients with chronic hepatitis C in (89.4%vs. 8.7%, p0.00), both with statistical significance; 3, expression and age of liver tissue of patients with chronic hepatitis B AIM2 (p=0.178), gender (p=0.148), e antigen (p=0.825) independent state; 4, with statistical significance and expression of ALT and AIM2 level (p=0.03), regardless of the level of AST (p=0.378); 5, HBV in high load group (HBV-DNA = 1 * 105copies/ml), the expression of AIM2 in HBV is higher than that of the low load group (HBV-DNA1 105 * copies/ml (P0.01) expression), both had statistical difference; 6, expression and liver inflammation grade AIM2 has high correlation (p=0.007), and fibrosis are not significantly related (p=0.101); 7, and the expression of caspase-1 in liver tissue of chronic hepatitis B AIM2 (rs= 0.738, P0.01), IL-1 beta (rs=0.527, P0.01) and IL-18 (rs=0.642, P0.01) expression was 姝ｇ浉鍏，

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