Ⅰ型干扰素系统的内源性激活在皮肌炎免疫病理机制中的作用研究

发布时间：2018-05-09 12:45

本文选题：特发性炎症性肌病 + 髓样树突状细胞　；参考：《山东大学》2015年博士论文

【摘要】：研究背景皮肌炎(dermatomyositis,DM)是一种公认的获得性骨骼肌自身免疫性疾病,为特发性炎症性肌病(idiopathic inflammatory myopathies, IIMs)的一个亚型,临床上以亚急性进展的近端肌无力、特征性皮疹以及病理上肌肉组织炎症细胞浸润和束周肌纤维萎缩为主要特征,到目前为止,其免疫病理机制尚未得到完全阐明。Ⅰ型干扰素(type Ⅰ interferon, IFN-I)在红斑狼疮、干燥综合征等自身免疫性疾病中发挥着重要作用。而今,Ⅰ型干扰素在特发性炎症性疾病固有免疫中的作用机制也越来越受到重视。能够分泌和产生大量Ⅰ型干扰素的浆细胞样树突状细胞(plasmacytoid dendritic cells, pDCs),以往的研究证实,广泛地分布于皮肌炎患者肌肉组织中的肌内膜和肌束膜内。而Ⅰ型干扰素的产生依赖于各种受体和信号因子,并通过外源性和内源性两种途径来诱导激活,外源性诱导途径主要见于病毒感染,由Toll样受体(Toll-like receptors, TLRs)识别相应的病毒核酸成分,而诱导Ⅰ型干扰素的产生；内源性激活途径主要由视黄酸诱导基因-Ⅰ(retinoic acidnducible gene I, RIG-I)介导,通过核酸与蛋白复合物(内源性自身免疫复合物),诱导生成Ⅰ型干扰素。TLRs和RIG-I作为IFN-I产生通路中的重要受体,在DM肌肉组织中是否同时表达,且以何种TLR表达为主,pDCs又通过何种机制产生大量的Ⅰ型干扰素,在国内外尚无报道。本课题通过免疫组化、双重免疫荧光、蛋白免疫印迹定量等实验方法,充分探讨TLRs和RIG-I在皮肌炎患者肌肉组织中内源性Ⅰ型干扰素生成中的作用机制。第一部分树突状细胞在特发性炎症性肌病中的分布特点目的分析树突状细胞在DM、多发性肌炎(polymyositis, PM)和包涵体肌炎(inclusion body myositis, IBM)患者肌肉组织中的分布特点,以初步探讨髓样树突状细胞(myeloid dendritic cell, mDCs)和pDCs在IIM发病中的免疫作用机制。方法总结分析进行肌肉活检的IIM患者27例,其中DM10例、PM10例、IBM4例和正常对照组3例。总结IIM患者的临床资料和病理学特点,所有活检肌肉组织均行苏木素-伊红染色(hematoxylin-eosin, HE)和血树突状细胞抗原1(blood dendritic cell antigen 1, BDCA-1)和BDCA-2免疫组织化学染色。结果mDCs在DM、PM和IBM患者的肌肉组织中的血管周围和炎性浸润处,均有明显的阳性表达。pDCs在DM中有明显的阳性表达,但在PM和IBM中呈现出无或仅有微弱的非特异性表达。10例DM患者中有8例患者可见到pDCs的显著浸润,主要分布在筋膜和宽大肌束膜内血管周围及肌内膜炎细胞浸润处。正常对照组未见mDCs和pDCs的阳性表达。结论树突状细胞可能参与IIM中炎性细胞浸润和肌纤维破坏,在IIM的免疫病理机制中发挥重要作用,尤其是pDCs在DM的特异性表达,提示pDCs与DM的特征性免疫病理密切相关。第二部分Toll样受体和视黄醇诱导基因-Ⅰ在皮肌炎免疫病理机制中的作用研究目的明确包括TLR-3、TLR-4、TLR-7和TLR-9在内的TLRs和RIG-I在皮肌炎中的表达特点,并结合TLRs和RIG-I的功能探讨其在皮肌炎免疫病理中的作用。方法选取行肌肉活检的患者31例,其中DM组20例,对照组11例,包括PM4例,面肩肱型肌营养不良(facioscapulohumeral muscular dystrophy, FSHD)4例和正常对照组3例；全部活检标本均行HE染色及TLR-3、TLR-4、TLR-7、TLR-9和RIG-I免疫组化染色,应用Western-blot定量检测肌肉组织中TLR-3、TLR-4、 TLR-7、TLR-9和RIG-I的表达含量。结果免疫组化染色显示TLR-3和RIG-I在DM组的束周萎缩处有明显的表达,而在DM和PM组的炎性浸润处和坏死再生纤维中,仅有轻微的非特异性表达,在FSHD组和正常对照组未见其表达,RIG-I在DM组筋膜中的炎性浸润处可见阳性表达；TLR-4和TLR-9在DM、PM和FSHD组的炎性浸润处均有明显的表达,而在正常对照组未见其表达,TLR-9在DM组筋膜中的炎性浸润处可见阳性表达；TLR-7仅在9例DM患者及少数PM患者的炎性浸润处可见的阳性表达,而在其他DM/PM中,以及FSHD的炎性浸润处未见表达,TLR-7在所有患者肌肉组织中的神经纤维上可见强阳性表达。Western-blot定量分析提示,TLR-3和RIG-I在DM中的表达明显上调,与各对照组相比具有统计学意义(p0.05); TLR-9在DM中的表达明显上调,与各对照组相比,具有显著的统计学意义(p0.05); TLR-4在DM、PM和FSHD各组患者肌肉中的的蛋白表达,之间比较无显著差异(p0.05), TLR-4在DM组的蛋白表达与正常对照组比较,存在统计学差异(p0.05); TLR-7在DM组中的表达上调,与其他对照组相比有统计学差异(p0.05)。结论RIG-I和TLR-3在DM中束周萎缩处的特异性分布,提示它们在DM的特征性免疫病理束周萎缩的形成过程中起重要作用。TLR-9在DM的肌肉组织中的表达含量明显上调,提示其在DM的免疫病理机制过程中有重要的参与作用。第三部分Toll样受体和视黄酸诱导基因-Ⅰ在皮肌炎患者肌肉组织中内源性Ⅰ型干扰素生成中的作用目的通过检测pDCs与TLR-3、TLR-4、TLR-7、TLR-9和RIG-I的共表达,以探讨DM患者肌肉组织中内源性IFN-I的生成机制。方法选取行肌肉活检的患者31例,其中DM组20例,对照组11例,包括PM4例,FSHD4例和正常对照组3例；全部活检标本均行TLR-3、TLR-4、TLR-7、TLR-9和RIG-I免疫荧光染色,用双重免疫荧光方法检测DM肌肉标本中浆细胞样树突状细胞(pDCs)与TLRs及RIG-I的共表达。结果免疫荧光染色显示TLR-3和RIG-I主要表达在DM组的束周萎缩处,而在DM和PM组的炎性浸润处和坏死再生纤维中,可见少量非特异性表达,在FSHD组和正常对照组未见其表达；TLR-4和TLR-9在DM、PM和FSHD组的炎性浸润处均有明显的表达,而在正常对照组未见其表达；TLR-7仅在7例DM患者及少数PM患者的炎性浸润处可见的阳性表达,而在其他DM/PM中,以及FSHD的炎性浸润处未见表达,TLR-7在所有患者肌肉组织中的神经纤维上可见强阳性表达；RIG-I和TLR-9在DM组筋膜中的炎性浸润处可见阳性表达。DM组的筋膜、肌内膜和肌束膜内可见大量pDCs与TLR-9的共表达细胞；在肌内膜中可见部分pDCs同时表达TLR-7。RIG-I和TLR-3虽然明显的表达在束周萎缩处,但与TLR.-4一样,均未发现与pDCs的共表达。结论TLR-9在DM肌肉组织中pDCs作用下的内源性IFN-I的生成中发挥主要途径作用,次要途经为TLR-7所介导。TLR-3和RIG-I选择性高表达在DM束周肌纤维上,提示其可能在一定程度上参与DM特征性病理特征——束周萎缩的形成。
[Abstract]:Background dermatomyositis (DM) is a recognized acquired autoimmune disease of skeletal muscle, a subtype of idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIMs), clinically with subacute progression of proximal myasthenia, characteristic rash, and pathological muscle tissue inflammatory cell infiltration and weeks. Muscle fiber atrophy is the main feature, so far, its immune pathological mechanism has not been fully elucidated. Type I interferon (type I interferon, IFN-I) plays an important role in autoimmune diseases such as lupus erythematosus, Sjogren syndrome and so on. More and more attention has been paid to the plasmacytoid dendritic cells (pDCs) that secretes and produces a large number of type I interferons. Previous studies have proved that it is widely distributed in the myomintima and the membrane of the muscle tissue of patients with dermatomyositis, and the production of type I interferon is dependent on various receptors and signal factors. Exogenous and endogenous two pathways are used to induce activation. Exogenous inducement pathway is mainly found in virus infection. The Toll like receptor (Toll-like receptors, TLRs) identifies the corresponding viral nucleic acid components and induces the production of type I interferon, and the endogenous activation pathway is mainly induced by retinoic acid (retinoic acidnducible gene I, RI). G-I) mediated, through nucleic acid and protein complex (endogenous autoimmunity complex), induced generation I interferon.TLRs and RIG-I as an important receptor in the IFN-I pathway, whether it is expressed at the same time in the DM muscle tissue, and which TLR expression is the dominant factor, and pDCs can produce a large number of type I interferon through the mechanism of pDCs, which is not yet available at home and abroad. In this study, the role of TLRs and RIG-I in the formation of endogenous interferon I in the muscle tissue of patients with dermatomyositis was investigated by immunohistochemistry, double immunofluorescence and immunoblotting. The distribution characteristics of dendritic cells in idiopathic inflammatory myopathy in part 1 were analyzed in order to analyze dendritic cells The distribution of muscle tissue in DM, polymyositis (PM) and inclusion body myositis (IBM), in order to preliminarily discuss the immune mechanism of myeloid dendritic cells (myeloid dendritic cell, mDCs) and pDCs in the pathogenesis of the disease. Methods 27 cases of muscle biopsy were summarized and analyzed. Cases, PM10 cases, IBM4 cases and 3 normal controls. The clinical data and pathological features of IIM patients were summarized. All the biopsy muscle tissues were treated with hematoxylin eosin staining (hematoxylin-eosin, HE) and blood dendritic cell antigen 1 (blood dendritic cell antigen 1, BDCA-1) and BDCA-2 immunohistochemical staining. The positive expression of.PDCs in DM was obviously expressed in the perivascular and inflammatory infiltration of the muscle tissue, but in PM and IBM, there were 8 cases of DM patients with no or only weak non specific expression of.10, which could see significant infiltration of pDCs, mainly in the perivascular and perivascular of the broad myofascial membrane and the broad myoplastic. There was no positive expression of mDCs and pDCs in the normal control group. Conclusion dendritic cells may participate in the infiltration of inflammatory cells and the damage of muscle fibers in IIM, and play an important role in the immuno pathological mechanism of IIM, especially the specific expression of pDCs in DM, suggesting that pDCs is closely related to the characteristic immune pathology of DM. Second parts are closely related to the immune pathology of DM. The role of Toll like receptor and retinol induced gene - I in the immuno pathological mechanism of dermatomyositis, the purpose of this study is to clarify the expression of TLRs and RIG-I in dermatomyositis, including TLR-3, TLR-4, TLR-7 and TLR-9, and to explore the role of TLRs and RIG-I in dermatomyositis. There were 31 cases, of which 20 cases in group DM and 11 cases in control group, including PM4, 4 cases of facioscapulohumeral muscular dystrophy, FSHD, and 3 cases of normal control group. All biopsy specimens were stained with HE staining and TLR-3, TLR-4, TLR-7, TLR-9 and RIG-I immunity. Results the expression of -7, TLR-9 and RIG-I showed that TLR-3 and RIG-I were clearly expressed in the peripheral atrophy of group DM, but in the inflammatory infiltration and necrotic fibers of the DM and PM groups, only a slight nonspecific expression was found in the group of FSHD and the normal control group, and RIG-I was in the inflammatory infiltration of the DM fascia. The positive expression of TLR-4 and TLR-9 was found in the inflammatory infiltration of DM, PM and FSHD groups, but no expression in the normal control group. TLR-9 was positive in the inflammatory infiltration of the fascia of the DM group; TLR-7 was only positive in the inflammatory infiltration of 9 cases of DM and a few PM patients, while in other DM/PM, and FS. There was no expression in the inflammatory infiltration of HD, and the strong positive expression of.Western-blot was found on the nerve fibers in all the muscle tissues of all the patients. The expression of TLR-3 and RIG-I in DM was obviously up - regulated, compared with the control group (P0.05), and the expression of TLR-9 in DM was obviously up - up. Compared with the control group, the expression of TLR-9 was significantly higher than that of the control groups. Statistical significance (P0.05); there was no significant difference in the expression of protein in the muscles of each group of DM, PM and FSHD (P0.05). The protein expression of TLR-4 in the DM group was compared with the normal control group, and there was a statistical difference (P0.05); TLR-7 in the DM group was up up, and compared with the other control groups, there were statistical differences (P0.05). Conclusions The specific distribution of G-I and TLR-3 in the peripheral atrophy of DM suggests that they play an important role in the formation of DM's characteristic peripheral atrophy, and the expression of.TLR-9 in the muscle tissue of DM is obviously up-regulated, suggesting that it plays an important role in the immune pathological mechanism of DM. The third part of Toll like receptor and retinol The role of acid induced gene - I in the production of endogenous interferon type I in the muscle tissue of patients with dermatomyositis, objective by detecting the co expression of pDCs and TLR-3, TLR-4, TLR-7, TLR-9 and RIG-I, in order to explore the mechanism of endogenous IFN-I in muscle tissue of DM patients. Methods 31 patients with muscle biopsy were selected, including 20 cases in DM group and 11 in control group. There were 3 cases of PM4, FSHD4 and normal control. All the biopsy specimens were performed TLR-3, TLR-4, TLR-7, TLR-9 and RIG-I immunofluorescence. The co expression of plasma like dendritic cells (pDCs) in DM muscle specimens was detected by double immunofluorescence, and the immunofluorescence staining showed that TLR-3 and main expression were mainly expressed in the group of DM muscles. In the inflammatory infiltration and necrotic fibers of group DM and PM, a small amount of nonspecific expression was found in group FSHD and normal control. TLR-4 and TLR-9 were clearly expressed in the inflammatory infiltration of DM, PM and FSHD groups, but no expression in the normal control group; TLR-7 was only in 7 cases of DM patients and a few PM patients. The positive expression of the inflammatory infiltration was seen in the other DM/PM, and the inflammatory infiltration of FSHD was not expressed. The strong positive expression of TLR-7 was found on the nerve fibers in all the muscle tissues of the patients, and RIG-I and TLR-9 were found positive in the fascia of the.DM group in the inflammatory infiltration of the fascia of the DM group, and the intima and myocutaneous membrane of the muscle were visible. The co expression of pDCs and TLR-9; partial pDCs was found in the intima, while TLR-7.RIG-I and TLR-3 were expressed in the peripheral atrophy, but no co expression with pDCs was found as TLR.-4. Conclusion TLR-9 plays a major role in the endogenous IFN-I production of pDCs in DM muscle tissue, and the secondary pathway is secondary. The selective high expression of.TLR-3 and RIG-I mediated by TLR-7 on the DM peri muscle fibers suggests that it may be involved in the characteristic pathological features of DM to a certain extent, the formation of peripheral atrophy.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R593.26

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