NLRP3及HMGB1在特发性炎性肌病发病机制中的作用及相关蛋白组学研究
发布时间：2018-07-13 15:52
【摘要】：研究背景:特发性炎性肌病(idiopathic inflamnmtorymyodtis,IIM )是一组自身免疫性疾病,其主要包括多发性肌炎(Polymyositis, PM)、皮肌炎(Dermatomyositis, DM)和包涵体肌炎。尽管许多证据表现IIM是由免疫介导所致,但是更具体的发病机制仍不是非常明了。研究表明,核苷酸结合寡聚化结构域受体蛋白3 (NOD-like receptor protein 3,NLRP3)与接头蛋白(凋亡相关的含有CARD区的斑片样蛋白,apoptosis-associated speck-like protein containing a CARD,ASC)和效应蛋白(半胱天冬酶-1,Caspase-1)组成的多蛋白复合体(NLRP3炎性小体)参与固有免疫应答,但NLRP3炎性小体是否与PM/DM患者肌肉组织中肌纤维变性、坏死相关,以及NLRP3在IIM发病过程中的主要作用,仍需进一步验证。高迁移率族蛋白1 (high mobility group box 1,HMGB1)作为一种重要的炎性物质在肺间质纤维化的动物模型中证实受caspase-l的调控与其下游物质IL-18和IL-1 β共同参与NLRP3炎性小体通路的致病过程,但在IIM中并无相关研究。蛋白组学广泛用于疾病研究,以寻找可能的某个疾病的生物标志物,为该疾病提供最佳的诊断金标准。许多相关研究提示IIM患者不论在血液还是在肌肉组织,极有可能存在的特殊相关的蛋白性生物标志物,且人们仍在努力探讨中。非定量蛋白质组学技术中的 SWATH-MS (Sequential Window Acquisition of all Theoretical fragment ions - Mass Spectrometry)是蛋白组学技术中比较稳定且应用较广的技术,具高通量等特点。在医学领域,SWATH-MS在临床诊断、发病机制及药物研发等方面有初步的应用成果。为此,采用SWATH-MS直接研究IIM患者病变的肌肉组织的蛋白组学非常有实际意义。研究目的:1、观察PM/DM患者及自身免疫性肌炎动物模型肌肉组织的NLRP3及HMGB1表达。2、通过免疫干预自身免疫性肌炎动物模型,观察NLRP3及HMGB1表达,以了解其治疗作用。3、通过PM/DM患者肌肉蛋白组学研究,以寻找可能的特发性炎性肌病的特异性蛋白标志物。材料与方法:1、选用12只BALB/c小鼠,采用豚鼠骨骼肌匀浆蛋白弗氏佐剂混悬液免疫方法制作本模型,观察动物的临床表现、血清CK含量及肌肉的病理变化。2、将18只BALB/c小鼠分为EAM治疗组、EAM组和对照组,采用免疫组织化学方法观察给予BAY 11-7082治疗后的动物模型临床表现和肌肉组织NLRP3与HMGB1的表达。3、采用免疫组织化学染色方法观察6例PM、10例DM和10例肌活检正常者(对照组)的肌肉组织NLRP3及HMGB1的表达。4、采用SWATH-MS技术检测DM、PM、免疫坏死性肌病和假肥大型肌营养不良(均各为3例)患者的肌肉组织蛋白组学,并将其结果通过网站对差异蛋白进行分析比较。研究结果:1、EAM组小鼠的临床表现明显,血清CK含量升高,病理为典型肌炎改变,提示本模型为理想的EAM动物模型。2、与EAM组和对照组比较,EAM治疗组临床表现显著好转(1.73±0.1 vs 1.55±0.1,P0.01),四肢肌力显著改善(11.0±0.7g/gvs8.1±0.2g/gvs13.4±0.7g/g,P0.01),肌肉组织NLRP3和HMGB1的mRNA及其蛋白表达均显著下降(P0.01)。3、与对照组比较,PM患者肌肉组织NLRP3表达均为阳性,DM患者肌肉组织,NLRP3的表达阳性率为80%; PM患者肌肉组织HMGB1均表达阳性,DM患者肌肉组织HMGB1的表达阳性率为30%。4、与DMD组比较,PM组检测出的差异蛋白有302种,DM组有484种;与NM组比较,PM组检测出的差异蛋白有176种,DM组有261种。为寻找与炎性免疫性相关的差异蛋白,进一步分析发现DM组有6种,PM组有10种与炎症相关。其中DM组的热休克蛋白90-β和双链糖蛋白与NLRP3炎性小体反应途径相关。PM组中细胞凋亡相关的含有CARD区的斑点样蛋白与NLRP3炎性小体反应途径相关。研究结论:1、NLRP3及HMGB1共同参与特发性炎性肌病的发病过程。2、通过干预NLRP3及HMGB1的表达,可能有效治疗效果。3、特发性炎性肌病可能存在与炎症相关的生物标志蛋白。
[Abstract]:Background: idiopathic inflamnmtorymyodtis (IIM) is a group of autoimmune diseases, which mainly include polymyositis (Polymyositis, PM), dermatomyositis (Dermatomyositis, DM) and inclusion body myositis. Although many evidence shows that IIM is caused by immunization, the more specific pathogenesis is still not very much. It is clear that nucleotides combine oligooligomeric domain receptor protein 3 (NOD-like receptor protein 3, NLRP3) with the joint protein (apoptosis related plaques containing CARD region, apoptosis-associated speck-like protein containing a CARD, ASC) and effect egg white (cystine asparaginase). NLRP3 inflammatory body participates in the inherent immune response, but it is still necessary to further verify whether the NLRP3 inflammatory corpuscle is related to muscle fibrosis, necrosis and the main role of NLRP3 in the pathogenesis of IIM in PM/DM patients. High mobility group protein 1 (high mobility group box 1, HMGB1) is an important inflammatory substance in the pulmonary interstitial In the animal model of fibrosis, it is confirmed that the regulation of caspase-l and its downstream substance IL-18 and IL-1 beta are involved in the pathogenesis of NLRP3 inflammatory small body pathway, but there is no related research in IIM. Proteomics is widely used in disease research to find biomarkers of a possible disease and provide the best diagnostic criteria for the disease. Many related studies suggest that IIM patients are highly likely to have specific related protein biomarkers, whether in blood or in muscle tissue, and that people are still trying to explore. The SWATH-MS (Sequential Window Acquisition of all Theoretical fragment ions - Mass Spectrometry) is an egg in the non quantitative proteomics technology. In the field of medicine, SWATH-MS has a preliminary application in the field of clinical diagnosis, pathogenesis and drug development. Therefore, it is of great practical significance to use SWATH-MS to study the egg white of the pathological muscle tissue of IIM patients. The purpose of this study is: 1. The expression of NLRP3 and HMGB1 in the muscle tissue of PM/DM patients and autoimmune myositis was.2. The expression of NLRP3 and HMGB1 was observed by immunizing autoimmune myositis and the expression of NLRP3 and HMGB1 was observed to understand the therapeutic effect of.3. The specific protein markers of idiopathic inflammatory myopathy were found through the study of the muscle protein group of PM/DM patients. Materials and methods: 1, 12 BALB/c mice were selected and the guinea pig's skeletal muscle homogenate protein Freund adjuvant was used to make this model. The clinical manifestations of the animals, the serum CK content and the pathological changes of the muscles were observed.2. 18 BALB/c mice were divided into EAM treatment group, EAM group and control group, and BAY 11 were observed by immunohistochemical method. The clinical manifestations of animal model after -7082 treatment and expression of NLRP3 and HMGB1 in muscle tissue were.3. Immunohistochemical staining was used to observe the expression of.4 in 6 cases of PM, 10 cases of DM and 10 cases of muscle biopsy (control group), NLRP3 and HMGB1, and SWATH-MS technique was used to detect DM, PM, immune necrotic myopathy and fake fat large muscular dystrophy. The muscle tissue proteomics of the 3 patients and the results were compared with the difference protein on the site. The results were as follows: 1, the clinical manifestations of the EAM group were obvious, the serum CK content increased and the pathological changes were typical myositis, suggesting that the model was the ideal EAM dynamic model.2, compared with the EAM group and the control group, the EAM treatment group was clinical. The muscle strength of the limbs was significantly improved (1.73 + 0.1 vs 1.55 + 0.1, P0.01), and the muscle strength of the extremities was significantly improved (11 + 0.7g/gvs8.1 + 0.2g/gvs13.4 + 0.7g/g, P0.01). The mRNA and protein expression of NLRP3 and HMGB1 in the muscle tissue decreased significantly (P0.01).3. The rate of sex was 80%, the expression of HMGB1 in the muscle tissue of PM patients was positive, and the positive rate of HMGB1 expression in the muscle tissue of DM patients was 30%.4. Compared with the DMD group, there were 302 different proteins in the PM group and 484 species in the DM group. Compared with the NM group, there were 176 differential proteins in the PM group and 261 in the DM group. One step analysis showed that there were 6 species in group DM, and 10 in group PM were associated with inflammation. In group DM, 90- beta and double chain glycoproteins associated with NLRP3 inflammatory small body reaction pathway associated with apoptotic cell apoptosis in the.PM group were associated with the pathway of NLRP3 inflammatory corpuscle. Conclusion: 1, NLRP3 and HMGB1 are involved in the idiopathic. The pathogenesis of inflammatory myopathy.2, by interfering with the expression of NLRP3 and HMGB1, may be effective in the treatment of.3. Idiopathic inflammatory myopathy may have a biomarker associated with inflammation.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.2
[Abstract]:Background: idiopathic inflamnmtorymyodtis (IIM) is a group of autoimmune diseases, which mainly include polymyositis (Polymyositis, PM), dermatomyositis (Dermatomyositis, DM) and inclusion body myositis. Although many evidence shows that IIM is caused by immunization, the more specific pathogenesis is still not very much. It is clear that nucleotides combine oligooligomeric domain receptor protein 3 (NOD-like receptor protein 3, NLRP3) with the joint protein (apoptosis related plaques containing CARD region, apoptosis-associated speck-like protein containing a CARD, ASC) and effect egg white (cystine asparaginase). NLRP3 inflammatory body participates in the inherent immune response, but it is still necessary to further verify whether the NLRP3 inflammatory corpuscle is related to muscle fibrosis, necrosis and the main role of NLRP3 in the pathogenesis of IIM in PM/DM patients. High mobility group protein 1 (high mobility group box 1, HMGB1) is an important inflammatory substance in the pulmonary interstitial In the animal model of fibrosis, it is confirmed that the regulation of caspase-l and its downstream substance IL-18 and IL-1 beta are involved in the pathogenesis of NLRP3 inflammatory small body pathway, but there is no related research in IIM. Proteomics is widely used in disease research to find biomarkers of a possible disease and provide the best diagnostic criteria for the disease. Many related studies suggest that IIM patients are highly likely to have specific related protein biomarkers, whether in blood or in muscle tissue, and that people are still trying to explore. The SWATH-MS (Sequential Window Acquisition of all Theoretical fragment ions - Mass Spectrometry) is an egg in the non quantitative proteomics technology. In the field of medicine, SWATH-MS has a preliminary application in the field of clinical diagnosis, pathogenesis and drug development. Therefore, it is of great practical significance to use SWATH-MS to study the egg white of the pathological muscle tissue of IIM patients. The purpose of this study is: 1. The expression of NLRP3 and HMGB1 in the muscle tissue of PM/DM patients and autoimmune myositis was.2. The expression of NLRP3 and HMGB1 was observed by immunizing autoimmune myositis and the expression of NLRP3 and HMGB1 was observed to understand the therapeutic effect of.3. The specific protein markers of idiopathic inflammatory myopathy were found through the study of the muscle protein group of PM/DM patients. Materials and methods: 1, 12 BALB/c mice were selected and the guinea pig's skeletal muscle homogenate protein Freund adjuvant was used to make this model. The clinical manifestations of the animals, the serum CK content and the pathological changes of the muscles were observed.2. 18 BALB/c mice were divided into EAM treatment group, EAM group and control group, and BAY 11 were observed by immunohistochemical method. The clinical manifestations of animal model after -7082 treatment and expression of NLRP3 and HMGB1 in muscle tissue were.3. Immunohistochemical staining was used to observe the expression of.4 in 6 cases of PM, 10 cases of DM and 10 cases of muscle biopsy (control group), NLRP3 and HMGB1, and SWATH-MS technique was used to detect DM, PM, immune necrotic myopathy and fake fat large muscular dystrophy. The muscle tissue proteomics of the 3 patients and the results were compared with the difference protein on the site. The results were as follows: 1, the clinical manifestations of the EAM group were obvious, the serum CK content increased and the pathological changes were typical myositis, suggesting that the model was the ideal EAM dynamic model.2, compared with the EAM group and the control group, the EAM treatment group was clinical. The muscle strength of the limbs was significantly improved (1.73 + 0.1 vs 1.55 + 0.1, P0.01), and the muscle strength of the extremities was significantly improved (11 + 0.7g/gvs8.1 + 0.2g/gvs13.4 + 0.7g/g, P0.01). The mRNA and protein expression of NLRP3 and HMGB1 in the muscle tissue decreased significantly (P0.01).3. The rate of sex was 80%, the expression of HMGB1 in the muscle tissue of PM patients was positive, and the positive rate of HMGB1 expression in the muscle tissue of DM patients was 30%.4. Compared with the DMD group, there were 302 different proteins in the PM group and 484 species in the DM group. Compared with the NM group, there were 176 differential proteins in the PM group and 261 in the DM group. One step analysis showed that there were 6 species in group DM, and 10 in group PM were associated with inflammation. In group DM, 90- beta and double chain glycoproteins associated with NLRP3 inflammatory small body reaction pathway associated with apoptotic cell apoptosis in the.PM group were associated with the pathway of NLRP3 inflammatory corpuscle. Conclusion: 1, NLRP3 and HMGB1 are involved in the idiopathic. The pathogenesis of inflammatory myopathy.2, by interfering with the expression of NLRP3 and HMGB1, may be effective in the treatment of.3. Idiopathic inflammatory myopathy may have a biomarker associated with inflammation.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.2
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