本文選題：慢性乙型肝炎　切入點(diǎn)：HBV相關(guān)性腎小球腎炎　出處：《山東大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：[背景/目的]HBV感染是個(gè)全球性問(wèn)題,由于HBV感染所導(dǎo)致的肝硬化,肝衰竭甚至肝癌等臨床結(jié)局嚴(yán)重的威脅人類的健康。同時(shí),HBV的感染也可導(dǎo)致許多肝外疾病的發(fā)生,HBV相關(guān)性腎小球腎炎即是常見(jiàn)的一種肝外HBV感染相關(guān)疾病。目前,關(guān)于兩種疾病的發(fā)病機(jī)制均不明確,大多數(shù)觀點(diǎn)認(rèn)為與免疫反應(yīng)相關(guān)。黑色素缺乏因子2 (AIM2)屬于HIN-200蛋白家族,其主要存在于細(xì)胞漿中,AIM2可被胞漿內(nèi)dsDNA所識(shí)別并激活,并結(jié)合中間蛋白ASC形成炎性復(fù)合體,進(jìn)一步激活caspase-1途徑,誘導(dǎo)IL-1β、IL-18等炎性因子的成熟和釋放,最終導(dǎo)致炎癥的發(fā)生,在機(jī)體的固有免疫反應(yīng)而誘發(fā)的炎癥中扮演重要作用。在慢性乙型肝炎中,乙肝病毒在復(fù)制的過(guò)程,首先位于肝細(xì)胞核內(nèi)的CCC-DNA為乙肝病毒復(fù)制的終模板,由其為模板合成負(fù)鏈DNA,再由負(fù)鏈DNA為模板合成正鏈DNA,然后形成ds-DNA,在由胞內(nèi)向胞外轉(zhuǎn)移的過(guò)程中,利用胞內(nèi)蛋白完成病毒的裝配而釋放。那么,在轉(zhuǎn)運(yùn)的過(guò)程中ds-DNA便有機(jī)會(huì)被胞漿內(nèi)的AIM2所識(shí)別,形成炎性復(fù)合體從而誘發(fā)肝臟炎癥;同樣,在HBV-GN中,既往已有研究報(bào)道電鏡檢測(cè)可以發(fā)現(xiàn)腎組織固有細(xì)胞內(nèi)存在有乙肝病毒樣顆粒及HBV-DNA的復(fù)制,那么腎臟固有細(xì)胞胞漿中的AIM2也有機(jī)會(huì)識(shí)別HBV-DNA并被激活,通過(guò)caspase-1途徑,釋放IL-1β、IL-18炎性因子,從而導(dǎo)致腎臟炎癥的發(fā)生。為了驗(yàn)證這一設(shè)想,我們進(jìn)行了此項(xiàng)研究,通過(guò)檢測(cè)肝臟及腎臟內(nèi)AIM2的表達(dá)及與caspase-1,IL-1 β及IL-18表達(dá)的關(guān)系,探討HBV與AIM2的激活在CHB、HBV-GN中可能的致病機(jī)制,為這一疾病的發(fā)病機(jī)制和治療提供新的思路和研究方法。[方法]研究通過(guò)組織免疫組化實(shí)驗(yàn)測(cè)定慢性乙型肝炎組織及HBV-GN的腎組織中AIM2水平,及其與caspase-1,IL-1 β、IL-18等炎性因子表達(dá)水平以及肝細(xì)胞,腎臟炎癥程度的關(guān)系。共計(jì)70例慢性肝炎的患者及79例慢性腎小球腎炎的患者納入到本項(xiàng)研究,其中在慢性肝炎患者中,慢性乙型肝炎47例為實(shí)驗(yàn)組,慢性丙肝患者23例為對(duì)照組;慢性腎小球腎炎的患者中,慢性乙肝相關(guān)性腎炎(HBV-GN)病人54例作為實(shí)驗(yàn)組,慢性腎小球腎炎(CGN)患者25例作為對(duì)照組。以上所有的病人均根據(jù)研究的要求行肝臟穿刺或腎臟穿刺取得肝或腎組織標(biāo)本制備石蠟包埋切片。應(yīng)用免疫組化法半定量測(cè)定患者肝、腎穿標(biāo)本中AIM2、Caspase-1、IL-18、IL-1p的表達(dá),同時(shí)分析AIM2的表達(dá)水平與臨床資料的相關(guān)性。本研究結(jié)果運(yùn)用SPSS 17.0軟件包進(jìn)行數(shù)據(jù)的統(tǒng)計(jì)學(xué)處理,兩組間AIM2的表達(dá)及臨床資料的的關(guān)系采用χ2檢驗(yàn)和Fisher精確概率檢驗(yàn),多個(gè)樣本間的單因素比較采用單因素方差分析,相關(guān)性分析采用Spearman's檢驗(yàn)。所有結(jié)果的分析均以P0.05為有統(tǒng)計(jì)學(xué)意義。[結(jié)果]1、在慢性乙型肝炎組織中,AIM2的表達(dá)主要定位于肝細(xì)胞胞漿內(nèi);2、AIM2在慢乙肝組織內(nèi)的表達(dá)明顯高于其在慢丙肝患者肝組織中的表達(dá)(89.4%vs. 8.7%,p0.00),兩者具有統(tǒng)計(jì)學(xué)意義;3、慢乙肝患者肝組織內(nèi)AIM2的表達(dá)與年齡(p=0.178)、性別(p=0.148)、e抗原的狀態(tài)(p=0.825)無(wú)關(guān);4、AIM2的表達(dá)與ALT的水平具有統(tǒng)計(jì)學(xué)意義(p=0.03),而與AST的水平無(wú)關(guān)(p=0.378);5、在HBV高載量組(HBV-DNA≥1×105copies/ml), AIM2的表達(dá)高于其在HBV低載量組(HBV-DNA1×105 copies/ml)的表達(dá)(p0.01),兩者具有統(tǒng)計(jì)學(xué)差異;6、AIM2的表達(dá)與肝臟炎癥分級(jí)存在高度的相關(guān)性(p=0.007),而和纖維化分級(jí)則無(wú)明顯相關(guān)(p=0.101);7、慢乙肝肝組織內(nèi) AIM2 的表達(dá)與 caspase-1(rs=0.738,p0.01),IL-1β(rs=0.527,p0.01)及 IL-18 (rs=0.642,p0.01)的表達(dá)呈正相關(guān);8、AIM2在HBV-GN腎組織中主要定位于腎小球系膜細(xì)胞及內(nèi)皮細(xì)胞內(nèi);9、在HBV-GN中,AIM2的陽(yáng)性表達(dá)率為81. 4%,明顯高于其在CGN中的表達(dá)(4.0%),兩者具有統(tǒng)計(jì)學(xué)差異(81.4% vs. 4.0%, p0. 01);10、AIM2的表達(dá)與血清e(cuò)抗原的狀態(tài)無(wú)關(guān)(p=0. 614);同時(shí)與腎組織中HBV相關(guān)抗原的沉積無(wú)關(guān)(p=0. 511);11、HBV-GN中高病毒載量組中AIM2的表達(dá)相比較于低病毒載量組有統(tǒng)計(jì)學(xué)差異(p0. 05);12、不同病理類型的HBV-GN中AIM2的表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(p=0. 940);13、HBV-GN腎組織中AIM2的表達(dá)分別與caspase-1的表達(dá)(rs = 0.444, p 0.01)以及IL-1β的表達(dá)(rs=0.379,p0.01)呈正相關(guān);而caspase-1的表達(dá)亦與IL-]β的表達(dá)呈正相關(guān)(rs = 0.515, p 0.01)。[結(jié)論]AIM2可能通過(guò)識(shí)別并結(jié)合HBV-DNA激活后活化Caspase-]形成炎性復(fù)合體,釋放致前炎性因子IL-1 β,IL-18造成CHB肝臟損傷及HBV-GN的腎損傷。
[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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