發(fā)布時(shí)間：2018-05-17 00:34

  本文選題：乙肝相關(guān)性腎小球腎炎 + 干擾素誘導(dǎo)蛋白16�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：[背景／目的] 乙肝病毒相關(guān)性腎小球腎炎(HBV-GN)是HBV感染主要的肝外表現(xiàn)之一,免疫復(fù)合物引起的腎組織損傷是HBV-GN目前較為公認(rèn)的發(fā)病機(jī)制。而現(xiàn)今免疫電鏡對(duì)HBV-GN腎組織的超微形態(tài)結(jié)構(gòu)觀察發(fā)現(xiàn)腎固有細(xì)胞內(nèi)HBV的存在,以及PT-PCR檢測(cè)技術(shù)在HBV-GN'腎組織內(nèi)直接檢測(cè)到HBV-DNA的復(fù)制,均提示除HBV抗原-抗體復(fù)合物在腎小球沉積等經(jīng)典免疫復(fù)合物致病途徑外,還應(yīng)該考慮HBV直接感染所致的組織損傷可能也參與了發(fā)病。據(jù)此有學(xué)者提出“HBV可在腎組織直接感染、復(fù)制及損傷”的觀點(diǎn)。雖有上述研究和證據(jù)支持這一觀點(diǎn),卻都僅為形態(tài)學(xué)觀察結(jié)果或分析推論,少有在分子水平闡明病毒的直接損傷機(jī)制的相關(guān)研究,因此仍需深入探討,以揭示HBV病毒本身在疾病發(fā)生的分子作用通路。 胞漿內(nèi)DNA是機(jī)體發(fā)生免疫應(yīng)答的一個(gè)強(qiáng)力激發(fā)因子,免疫反應(yīng)過(guò)程中所涉及的下游信號(hào)通道已被廣泛研究和揭示,但是機(jī)體對(duì)細(xì)胞漿內(nèi)的外源性DNA的識(shí)別和觸發(fā)機(jī)制知之甚少。近年來(lái)免疫學(xué)基礎(chǔ)研究發(fā)現(xiàn)：PYHIN蛋白成員--IFI16和AIM2是細(xì)胞內(nèi)的雙鏈DNA感受器,IFI16能夠感受胞質(zhì)和核內(nèi)dsDNA,發(fā)起天然免疫應(yīng)答,通過(guò)不同信號(hào)通路最終誘導(dǎo)產(chǎn)生IFN-β參與炎癥反應(yīng)；AIM2可感知轉(zhuǎn)染到胞質(zhì)中的外源性dsDNA并與其結(jié)合,通過(guò)其特殊結(jié)構(gòu)域使Caspas-1的活化,形成炎性復(fù)合體,參與炎癥反應(yīng)。不同研究證明,細(xì)胞感染病毒后,細(xì)胞內(nèi)的外源性dsDNA會(huì)激活A(yù)IM2并使之持續(xù)表達(dá),成為一個(gè)重要的炎癥反應(yīng)物并觸發(fā)一系列胞內(nèi)反應(yīng),分泌和釋放IL-18、IL-1β,進(jìn)而發(fā)揮致炎和致凋亡等多種重要的生物學(xué)活性。其中由TLR4介導(dǎo)的信號(hào)通路中MyD88依賴性途徑,通過(guò)TIR區(qū)域向胞內(nèi)傳遞信號(hào),激活JNK和NF-κB等因子,引起[L家族蛋白及I FN的釋放這一下游信號(hào)通路已被多項(xiàng)研究證實(shí)。 近年來(lái)陸續(xù)可見(jiàn)關(guān)于PYHIN家族蛋白及細(xì)胞內(nèi)DNA感受器的研究報(bào)道,但均局限于免疫學(xué)基礎(chǔ)研究及腫瘤病因?qū)W研究,在感染性疾病中的研究報(bào)道則主要集中在細(xì)菌感染方面,少量關(guān)于HBV與AIM2的研究主要為基礎(chǔ)研究,未見(jiàn)PYHIN家族蛋白在HBV感染性疾病中的研究和探討。HBV是雙鏈DNA病毒,基于在HBV-GN腎組織腎小球固有細(xì)胞內(nèi)中已經(jīng)明確發(fā)現(xiàn)HBV-GN存在及其復(fù)制,結(jié)合細(xì)胞內(nèi)IFI16和AIM2相關(guān)研究取得的上述進(jìn)展,本研究設(shè)定IFI16. AIM2為研究目標(biāo),將其能夠感知細(xì)胞內(nèi)dsDNA后發(fā)生一系列致炎作用這一特性應(yīng)用于HBV-GN的發(fā)病機(jī)制的研究中,探討HBV病毒DNA在腎組織中是否激活細(xì)胞內(nèi)IFI16、AIM2,及其活化后在HBV-GN這一臨床疾病中的具體致病機(jī)制。同時(shí)明確作用受體,使IFI16、AIM2炎性復(fù)合體成為治療HBV-GN等病毒等感染性疾病的一個(gè)可能的目標(biāo),為疾病的臨床治療和藥物的開(kāi)發(fā)設(shè)計(jì)提供新的思路。 [方法] 本研究運(yùn)用組織學(xué)實(shí)驗(yàn)結(jié)合體外細(xì)胞學(xué)實(shí)驗(yàn)兩條路線進(jìn)行,相互印證。應(yīng)用腎穿刺組織活檢常規(guī)病理技術(shù)(包括HE染色及特殊染色)、免疫熒光染色、免疫組織化學(xué)染色及電鏡技術(shù),結(jié)合臨床檢查明確HBV-GN診斷,同時(shí)應(yīng)用電子顯微鏡技術(shù)篩選腎組織內(nèi)存在HBV病毒顆粒的HBV-GN病例入組(實(shí)驗(yàn)組：45例),以原發(fā)性膜性腎病病例為對(duì)照組。應(yīng)用免疫組化染色檢測(cè)組織內(nèi)IFI16、IFN-β、 AIM2、Caspase-1、IL-1β、IL-18的表達(dá)情況,明確腎組織內(nèi)HBV感染與IFI16、AIM2的表達(dá)之間的相關(guān)性,明確IFI16的激活與IFN-β的表達(dá)情況、AIM2的激活與Caspase-1的表達(dá)及其下游因子IL-1β、IL-18的表達(dá)情況的相關(guān)性,及IFU16和AIM2表達(dá)之間的相關(guān)性。 將腎小球固有細(xì)胞(包括內(nèi)皮細(xì)胞HREC、系膜細(xì)胞HRMC.足細(xì)胞HRPC)進(jìn)行體外培養(yǎng),經(jīng)pcDNA3.0-1.1HBV轉(zhuǎn)染后,分別進(jìn)行PT-PCR檢測(cè)及Western Blot檢測(cè),定位及定量測(cè)定受HBV感染的腎小球固有細(xì)胞胞漿中各項(xiàng)指標(biāo)(IFI16、AIM2、IFN-β、Caspase-1、IL-1β和IL-18) mRNA及蛋白產(chǎn)物的表達(dá)水平,與非轉(zhuǎn)染組對(duì)照,分析相關(guān)性,探討HBV-DNA在觸發(fā)IFI16、AIM2的激活與表達(dá)后,IFI16與IFN-β、AIM2與Caspase-1激活之間的關(guān)系,炎性復(fù)合體的形成,以及與下游炎癥因子IL-1β、IL-18分泌與釋放之間的相關(guān)性。 細(xì)胞實(shí)驗(yàn)中,分別構(gòu)建工FI16和AIM2的siRNA沉默基因,設(shè)立siRNA與HBV共轉(zhuǎn)染組,在同樣存在HBV感染條件下重復(fù)測(cè)定各系細(xì)胞內(nèi)IFI16、IFN-β、AIM2、 Caspase-1以及下游因子的含量,分析兩兩之間相關(guān)性,在同一實(shí)驗(yàn)路線中雙向進(jìn)行驗(yàn)證,明確HBV-DNA激活I(lǐng)FI16、AIM2是病毒直接損傷作用的關(guān)鍵觸發(fā)因素,炎性復(fù)合體形成并導(dǎo)致炎性損傷在HBV-GN發(fā)病中發(fā)揮重要作用。兩條路線相互印證,互為補(bǔ)充。 [結(jié)果] 電鏡檢測(cè)在19.07%HBV-GN患者腎組織內(nèi)發(fā)現(xiàn)HBV病毒樣顆粒,明確了HBV在肝外腎組織內(nèi)的沉積。另外發(fā)現(xiàn)少數(shù)組織學(xué)檢查HBsAg、HBcAg均為陰性、按照診斷標(biāo)準(zhǔn)無(wú)法診斷為HBV-GN的HBV患者,在腎組織內(nèi)查到病毒樣顆粒,這種情況下電鏡檢查可以作為補(bǔ)充手段補(bǔ)足診斷依據(jù),修正補(bǔ)充光鏡診斷,減少漏診或誤診。 HBV-GN臨床表現(xiàn)復(fù)雜,最常出現(xiàn)的腎損害表現(xiàn)為蛋白尿,其次為血尿合并蛋白尿或腎病綜合癥。病理形態(tài)學(xué)表現(xiàn)多種多樣,以膜性腎病、膜性腎病伴非典型性改變及膜增生性腎小球腎炎改變最為多見(jiàn)；平均發(fā)病年齡：36.5±12.5歲；男性多于女性。 HBV-GN組腎組織中IFI16、AIM2的表達(dá)顯著高于原發(fā)性膜性腎病組(對(duì)照組),說(shuō)明IFI16和AIM2的激活與HBV感染關(guān)系密切。其中IFI16的表達(dá)強(qiáng)度與IFN-β的表達(dá)強(qiáng)度呈正相關(guān),AIM2表達(dá)強(qiáng)度與Caspase-1的表達(dá)強(qiáng)度呈正相關(guān),IL-1β、IL-18的表達(dá)強(qiáng)度與Caspase-1的表達(dá)強(qiáng)度呈正相關(guān)。 三系細(xì)胞中IFI16、IFN-β、AIM2、Caspase-1、IL-1β、IL-18的mRNA和蛋白水平的檢測(cè)結(jié)果顯示：與對(duì)照組相比,pcDNA3-3HBV轉(zhuǎn)染組IFI16、IFN-β、 AIM2、Caspase-1、IL-1β、IL-18的mRNA水平和蛋白表達(dá)量均明顯增加,其中IFI16的表達(dá)強(qiáng)度與IFN-β的表達(dá)強(qiáng)度呈顯著正相關(guān),AIM2的表達(dá)與Caspase-1、 IL-1β、IL-18的表達(dá)密切相關(guān)；siRNA和HBV共轉(zhuǎn)染組與單純HBV轉(zhuǎn)染組相比,所有檢測(cè)指標(biāo)均明顯降低,其中沉默IFI16基因可使IFN-β表達(dá)量明顯下降；沉默AIM2基因后,Caspase-1, IL-1β,IL-18表達(dá)量明顯下降。RT-PCR及Western blot檢測(cè)結(jié)果一致。 HBV-GN腎組織免疫組化實(shí)驗(yàn)結(jié)果與體外細(xì)胞實(shí)驗(yàn)結(jié)果一致。 [結(jié)論] HBV-GN中,PYHIN家族成員中的兩個(gè)蛋白--IFI16、A1M2為細(xì)胞內(nèi)外源性DNA感受器,可感知胞內(nèi)HBV DNA,與外源性dsDNA結(jié)合導(dǎo)致二者活化后,通過(guò)不同作用通路參與了HBV-GN的發(fā)生：1.IF116激活后主要通過(guò)誘導(dǎo)產(chǎn)生致炎因子IFN-β;2.AIM2激活后活化Caspase-1并與其結(jié)合形成炎性復(fù)合體,釋放致炎因子IL-1β, IL-18。研究證實(shí)除經(jīng)典免疫復(fù)合物損傷途徑外,HBV在腎組織內(nèi)的感染及復(fù)制,經(jīng)細(xì)胞內(nèi)DNA感受器識(shí)別后激活機(jī)體固有免疫反應(yīng),導(dǎo)致組織損傷。
[Abstract]:[background / purpose]
Hepatitis B virus associated glomerulonephritis (HBV-GN) is one of the major extrahepatic manifestations of HBV infection. The renal tissue damage caused by immune complex is a relatively recognized pathogenesis of HBV-GN. The ultrastructural observation of HBV-GN renal tissue by immuno electron microscopy today found the existence of HBV in the renal innate cells and the PT-PCR detection technique in HBV The direct detection of the replication of HBV-DNA in the -GN'renal tissue suggests that the tissue damage caused by the direct HBV infection may also be involved in the pathogenesis of the classical immune complex, such as the HBV antigen antibody complex in the glomeruli deposition, and some scholars suggest that "HBV can be directly infected, replicated and damaged in the renal tissue". Point. Although the above research and evidence support this view, it is only a morphological observation or analysis inference, few studies on the mechanism of direct damage to the virus at the molecular level, so it is still necessary to explore the HBV virus itself in the molecular pathway of the disease.
Intracellular DNA is a powerful stimulating factor in the immune response of the body. The downstream signal pathway involved in the immune response has been widely studied and revealed, but the body's recognition and triggering mechanism for exogenous DNA in the cytoplasm is little known. In recent years, the basic immunological study found that the members of the PYHIN protein, --IFI16 and AIM2, are the most important factors in the immunological process. The double stranded DNA receptor in the cell, IFI16 can feel the cytoplasm and the dsDNA in the nucleus, initiates the natural immune response, and induces the IFN- beta to participate in the inflammatory reaction through different signal pathways; AIM2 can perceive the exogenous dsDNA transfected into the cytoplasm and combine with it, and form an inflammatory complex through its special structure domain to activate the Caspas-1, and form an inflammatory complex. In response to inflammation, different studies have shown that after cells infect the virus, the exogenous dsDNA in the cell activates AIM2 and makes it continuous expression, becomes an important inflammatory reaction and triggers a series of intracellular reactions, secretes and releases IL-18, IL-1 beta, and then plays a variety of important biological activities, such as inflammation and apoptosis, which is mediated by TLR4 The MyD88 dependent pathway in the signal pathway, transmits signals through the TIR region to the intracellular signal, activates the factors such as JNK and NF- kappa B, causing the release of the [L family protein and I FN. The downstream signal pathway has been confirmed by a number of studies.
In recent years, research reports on PYHIN family proteins and intracellular DNA receptors have been seen, but they are limited to basic immunology and oncology. The research reports on infectious diseases are mainly focused on bacterial infection. A small number of studies on HBV and AIM2 are mainly based on basic research, and no PYHIN family protein is in HB The study and discussion of.HBV in V infectious diseases is a double stranded DNA virus. Based on the clear discovery of the existence and replication of HBV-GN in the renal glomerular innate cells of the renal tissue of HBV-GN, combined with the advances in the intracellular IFI16 and AIM2 related studies, this study sets IFI16. AIM2 as a research target to be able to perceive the occurrence of intracellular dsDNA. A series of inflammatory properties are applied to the study of the pathogenesis of HBV-GN, to explore whether the HBV virus DNA activates IFI16, AIM2, and its specific pathogenesis in the clinical disease of HBV-GN after activation in the renal tissue. Meanwhile, the receptor is clearly used to make the IFI16, AIM2 inflammatory complex become a treatment for the infection of the HBV-GN and other viruses. A possible goal of sexual diseases is to provide new ideas for clinical treatment of diseases and development and design of drugs.
[method]
This study was carried out with two routes of histology and in vitro cytology experiment. The routine pathological techniques of renal biopsy (including HE staining and special staining), immunofluorescence staining, immunohistochemical staining and electron microscopy, combined with clinical examination, HBV-GN diagnosis, and electronic microscope technical screening were used. The HBV virus particles in the renal tissue were included in the group of HBV-GN cases (experimental group: 45 cases), with primary membranous nephropathy as the control group. Immunohistochemical staining was used to detect the expression of IFI16, IFN- beta, AIM2, Caspase-1, IL-1 beta, IL-18, and the correlation between HBV infection in the renal tissue and IFI16, AIM2 expression in the renal tissue, and the IFI16 was clearly defined. Activation and expression of IFN- beta, the activation of AIM2 and the expression of Caspase-1, the correlation of the downstream factor IL-1 beta, the expression of IL-18, and the correlation between IFU16 and AIM2 expression.
The glomerular mesangial cells (including endothelial cell HREC, mesangial cell HRMC. HRPC) were cultured in vitro. After pcDNA3.0-1.1HBV transfection, PT-PCR detection and Western Blot detection were carried out to locate and quantify each item in the cytoplasm of the glomerular innate cells infected by HBV (IFI16, AIM2, IFN- beta, Caspase-1, beta and pcDNA3.0-1.1HBV) And the expression level of protein products, compared with the non transfection group, analyzed the correlation, and explored the relationship between the activation and expression of IFI16 and AIM2, the relationship between IFI16 and IFN- beta, AIM2 and Caspase-1 activation, the formation of inflammatory complex, and the correlation with the downstream inflammatory factor IL-1 beta, IL-18 secretion and release.
In the cell experiment, the siRNA silencing genes of FI16 and AIM2 were constructed, and the siRNA and HBV co transfection groups were set up, and the contents of IFI16, IFN- beta, AIM2, Caspase-1 and downstream factors in the cells of each line were repeated under the same HBV infection conditions, and the correlation between 22 was analyzed. A two-way verification was carried out in the same experimental route, and HBV-DNA activation was made. IFI16, AIM2 is the key trigger factor for the direct damage of the virus. The formation of the inflammatory complex and the cause of inflammatory damage play an important role in the pathogenesis of HBV-GN. The two routes are mutually confirmed and complementary to each other.
[results]
HBV virus like particles were found in the renal tissue of 19.07%HBV-GN patients by electron microscopy, and the deposition of HBV in the liver and kidney tissues was identified. In addition, a few histologically examined HBsAg, HBcAg were negative, and the HBV patients who were not diagnosed as HBV-GN were diagnosed with the diagnostic criteria, and the disease like particles were found in the renal tissue. In this case, the electron microscope examination could be used as a case. Supplementary means complement the diagnostic basis, modify the supplementary light microscopic diagnosis, reduce missed diagnosis or misdiagnosis.
The most common clinical manifestations of HBV-GN were proteinuria, followed by hematuria with proteinuria or nephrotic syndrome. The pathomorphologic manifestations were varied, with membranous nephropathy, membranous nephropathy accompanied by atypical change and membranous proliferative glomerulonephritis, with an average age of 36.5 + 12.5 years; more males In women.
The expression of IFI16 and AIM2 in the renal tissue of group HBV-GN was significantly higher than that of the primary membranous nephropathy group (control group), indicating that the activation of IFI16 and AIM2 was closely related to HBV infection. The expression intensity of IFI16 was positively correlated with the expression intensity of IFN- beta, and the intensity of AIM2 expression was positively correlated with the intensity of Caspase-1 expression, IL-1 beta, and the expression intensity of IL-18. The intensity of expression is positively correlated.
The results of the detection of mRNA and protein levels of IFI16, IFN- beta, AIM2, Caspase-1, IL-1 beta and IL-18 in three line cells showed that the levels of IFI16, IFN- beta, AIM2, Caspase-1, and protein in the pcDNA3-3HBV transfected group were significantly increased, and the expression intensity was significantly positively correlated with the expression intensity of the beta cells. The expression of M2 was closely related to the expression of Caspase-1, IL-1 beta and IL-18; all the detection indexes of siRNA and HBV co transfected groups were significantly lower than those of pure HBV transfection group, and the silent IFI16 gene could reduce the IFN- beta expression significantly, and the Caspase-1, IL-1 beta, and IL-1 beta, after the silencing of the AIM2 gene, were significantly reduced and detected. The result is consistent.
The immunohistochemical results of HBV-GN renal tissue were in accordance with the results of in vitro cell experiments.
[Conclusion]
In HBV-GN, two protein --IFI16 in PYHIN family members, A1M2 as endogenous DNA receptor, can perceive intracellular HBV DNA. After activation with exogenous dsDNA, it participates in the occurrence of HBV-GN through different pathways: 1.IF116 activation is mainly induced to produce inflammatory factor IFN- beta; 2.AIM2 activates after activation. In addition to the combination of the inflammatory complex and releasing inflammatory factor IL-1 beta, IL-18. studies confirmed that the infection and replication of HBV in the renal tissue except the classical immune complex damage pathway, and the intrinsic immune response of the body after the identification of the intracellular DNA receptor, resulting in tissue damage.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.31;R512.62

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 許勇芝;唐德q，

本文編號(hào)：1899121