PYHIN家族蛋白感知肾固有细胞内HBV DNA参与乙肝病毒相关性肾炎发病机制的研究

发布时间：2018-05-17 00:34

本文选题：乙肝相关性肾小球肾炎 + 干扰素诱导蛋白16　；参考：《山东大学》2014年博士论文

【摘要】：[背景／目的] 乙肝病毒相关性肾小球肾炎(HBV-GN)是HBV感染主要的肝外表现之一,免疫复合物引起的肾组织损伤是HBV-GN目前较为公认的发病机制。而现今免疫电镜对HBV-GN肾组织的超微形态结构观察发现肾固有细胞内HBV的存在,以及PT-PCR检测技术在HBV-GN'肾组织内直接检测到HBV-DNA的复制,均提示除HBV抗原-抗体复合物在肾小球沉积等经典免疫复合物致病途径外,还应该考虑HBV直接感染所致的组织损伤可能也参与了发病。据此有学者提出“HBV可在肾组织直接感染、复制及损伤”的观点。虽有上述研究和证据支持这一观点,却都仅为形态学观察结果或分析推论,少有在分子水平阐明病毒的直接损伤机制的相关研究,因此仍需深入探讨,以揭示HBV病毒本身在疾病发生的分子作用通路。胞浆内DNA是机体发生免疫应答的一个强力激发因子,免疫反应过程中所涉及的下游信号通道已被广泛研究和揭示,但是机体对细胞浆内的外源性DNA的识别和触发机制知之甚少。近年来免疫学基础研究发现：PYHIN蛋白成员--IFI16和AIM2是细胞内的双链DNA感受器,IFI16能够感受胞质和核内dsDNA,发起天然免疫应答,通过不同信号通路最终诱导产生IFN-β参与炎症反应；AIM2可感知转染到胞质中的外源性dsDNA并与其结合,通过其特殊结构域使Caspas-1的活化,形成炎性复合体,参与炎症反应。不同研究证明,细胞感染病毒后,细胞内的外源性dsDNA会激活AIM2并使之持续表达,成为一个重要的炎症反应物并触发一系列胞内反应,分泌和释放IL-18、IL-1β,进而发挥致炎和致凋亡等多种重要的生物学活性。其中由TLR4介导的信号通路中MyD88依赖性途径,通过TIR区域向胞内传递信号,激活JNK和NF-κB等因子,引起[L家族蛋白及I FN的释放这一下游信号通路已被多项研究证实。近年来陆续可见关于PYHIN家族蛋白及细胞内DNA感受器的研究报道,但均局限于免疫学基础研究及肿瘤病因学研究,在感染性疾病中的研究报道则主要集中在细菌感染方面,少量关于HBV与AIM2的研究主要为基础研究,未见PYHIN家族蛋白在HBV感染性疾病中的研究和探讨。HBV是双链DNA病毒,基于在HBV-GN肾组织肾小球固有细胞内中已经明确发现HBV-GN存在及其复制,结合细胞内IFI16和AIM2相关研究取得的上述进展,本研究设定IFI16. AIM2为研究目标,将其能够感知细胞内dsDNA后发生一系列致炎作用这一特性应用于HBV-GN的发病机制的研究中,探讨HBV病毒DNA在肾组织中是否激活细胞内IFI16、AIM2,及其活化后在HBV-GN这一临床疾病中的具体致病机制。同时明确作用受体,使IFI16、AIM2炎性复合体成为治疗HBV-GN等病毒等感染性疾病的一个可能的目标,为疾病的临床治疗和药物的开发设计提供新的思路。 [方法] 本研究运用组织学实验结合体外细胞学实验两条路线进行,相互印证。应用肾穿刺组织活检常规病理技术(包括HE染色及特殊染色)、免疫荧光染色、免疫组织化学染色及电镜技术,结合临床检查明确HBV-GN诊断,同时应用电子显微镜技术筛选肾组织内存在HBV病毒颗粒的HBV-GN病例入组(实验组：45例),以原发性膜性肾病病例为对照组。应用免疫组化染色检测组织内IFI16、IFN-β、 AIM2、Caspase-1、IL-1β、IL-18的表达情况,明确肾组织内HBV感染与IFI16、AIM2的表达之间的相关性,明确IFI16的激活与IFN-β的表达情况、AIM2的激活与Caspase-1的表达及其下游因子IL-1β、IL-18的表达情况的相关性,及IFU16和AIM2表达之间的相关性。将肾小球固有细胞(包括内皮细胞HREC、系膜细胞HRMC.足细胞HRPC)进行体外培养,经pcDNA3.0-1.1HBV转染后,分别进行PT-PCR检测及Western Blot检测,定位及定量测定受HBV感染的肾小球固有细胞胞浆中各项指标(IFI16、AIM2、IFN-β、Caspase-1、IL-1β和IL-18) mRNA及蛋白产物的表达水平,与非转染组对照,分析相关性,探讨HBV-DNA在触发IFI16、AIM2的激活与表达后,IFI16与IFN-β、AIM2与Caspase-1激活之间的关系,炎性复合体的形成,以及与下游炎症因子IL-1β、IL-18分泌与释放之间的相关性。细胞实验中,分别构建工FI16和AIM2的siRNA沉默基因,设立siRNA与HBV共转染组,在同样存在HBV感染条件下重复测定各系细胞内IFI16、IFN-β、AIM2、 Caspase-1以及下游因子的含量,分析两两之间相关性,在同一实验路线中双向进行验证,明确HBV-DNA激活IFI16、AIM2是病毒直接损伤作用的关键触发因素,炎性复合体形成并导致炎性损伤在HBV-GN发病中发挥重要作用。两条路线相互印证,互为补充。 [结果] 电镜检测在19.07%HBV-GN患者肾组织内发现HBV病毒样颗粒,明确了HBV在肝外肾组织内的沉积。另外发现少数组织学检查HBsAg、HBcAg均为阴性、按照诊断标准无法诊断为HBV-GN的HBV患者,在肾组织内查到病毒样颗粒,这种情况下电镜检查可以作为补充手段补足诊断依据,修正补充光镜诊断,减少漏诊或误诊。 HBV-GN临床表现复杂,最常出现的肾损害表现为蛋白尿,其次为血尿合并蛋白尿或肾病综合症。病理形态学表现多种多样,以膜性肾病、膜性肾病伴非典型性改变及膜增生性肾小球肾炎改变最为多见；平均发病年龄：36.5±12.5岁；男性多于女性。 HBV-GN组肾组织中IFI16、AIM2的表达显著高于原发性膜性肾病组(对照组),说明IFI16和AIM2的激活与HBV感染关系密切。其中IFI16的表达强度与IFN-β的表达强度呈正相关,AIM2表达强度与Caspase-1的表达强度呈正相关,IL-1β、IL-18的表达强度与Caspase-1的表达强度呈正相关。三系细胞中IFI16、IFN-β、AIM2、Caspase-1、IL-1β、IL-18的mRNA和蛋白水平的检测结果显示：与对照组相比,pcDNA3-3HBV转染组IFI16、IFN-β、 AIM2、Caspase-1、IL-1β、IL-18的mRNA水平和蛋白表达量均明显增加,其中IFI16的表达强度与IFN-β的表达强度呈显著正相关,AIM2的表达与Caspase-1、 IL-1β、IL-18的表达密切相关；siRNA和HBV共转染组与单纯HBV转染组相比,所有检测指标均明显降低,其中沉默IFI16基因可使IFN-β表达量明显下降；沉默AIM2基因后,Caspase-1, IL-1β,IL-18表达量明显下降。RT-PCR及Western blot检测结果一致。 HBV-GN肾组织免疫组化实验结果与体外细胞实验结果一致。 [结论] HBV-GN中,PYHIN家族成员中的两个蛋白--IFI16、A1M2为细胞内外源性DNA感受器,可感知胞内HBV DNA,与外源性dsDNA结合导致二者活化后,通过不同作用通路参与了HBV-GN的发生：1.IF116激活后主要通过诱导产生致炎因子IFN-β;2.AIM2激活后活化Caspase-1并与其结合形成炎性复合体,释放致炎因子IL-1β, IL-18。研究证实除经典免疫复合物损伤途径外,HBV在肾组织内的感染及复制,经细胞内DNA感受器识别后激活机体固有免疫反应,导致组织损伤。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.31;R512.62

【参考文献】