【摘要】：研究背景:特發(fā)性炎性肌病(idiopathic inflamnmtorymyodtis,IIM )是一組自身免疫性疾病,其主要包括多發(fā)性肌炎(Polymyositis, PM)、皮肌炎(Dermatomyositis, DM)和包涵體肌炎。盡管許多證據(jù)表現(xiàn)IIM是由免疫介導所致,但是更具體的發(fā)病機制仍不是非常明了。研究表明,核苷酸結(jié)合寡聚化結(jié)構(gòu)域受體蛋白3 (NOD-like receptor protein 3,NLRP3)與接頭蛋白(凋亡相關的含有CARD區(qū)的斑片樣蛋白,apoptosis-associated speck-like protein containing a CARD,ASC)和效應蛋白(半胱天冬酶-1,Caspase-1)組成的多蛋白復合體(NLRP3炎性小體)參與固有免疫應答,但NLRP3炎性小體是否與PM/DM患者肌肉組織中肌纖維變性、壞死相關,以及NLRP3在IIM發(fā)病過程中的主要作用,仍需進一步驗證。高遷移率族蛋白1 (high mobility group box 1,HMGB1)作為一種重要的炎性物質(zhì)在肺間質(zhì)纖維化的動物模型中證實受caspase-l的調(diào)控與其下游物質(zhì)IL-18和IL-1 β共同參與NLRP3炎性小體通路的致病過程,但在IIM中并無相關研究。蛋白組學廣泛用于疾病研究,以尋找可能的某個疾病的生物標志物,為該疾病提供最佳的診斷金標準。許多相關研究提示IIM患者不論在血液還是在肌肉組織,極有可能存在的特殊相關的蛋白性生物標志物,且人們?nèi)栽谂μ接懼小７嵌康鞍踪|(zhì)組學技術中的 SWATH-MS (Sequential Window Acquisition of all Theoretical fragment ions - Mass Spectrometry)是蛋白組學技術中比較穩(wěn)定且應用較廣的技術,具高通量等特點。在醫(yī)學領域,SWATH-MS在臨床診斷、發(fā)病機制及藥物研發(fā)等方面有初步的應用成果。為此,采用SWATH-MS直接研究IIM患者病變的肌肉組織的蛋白組學非常有實際意義。研究目的:1、觀察PM/DM患者及自身免疫性肌炎動物模型肌肉組織的NLRP3及HMGB1表達。2、通過免疫干預自身免疫性肌炎動物模型,觀察NLRP3及HMGB1表達,以了解其治療作用。3、通過PM/DM患者肌肉蛋白組學研究,以尋找可能的特發(fā)性炎性肌病的特異性蛋白標志物。材料與方法:1、選用12只BALB/c小鼠,采用豚鼠骨骼肌勻漿蛋白弗氏佐劑混懸液免疫方法制作本模型,觀察動物的臨床表現(xiàn)、血清CK含量及肌肉的病理變化。2、將18只BALB/c小鼠分為EAM治療組、EAM組和對照組,采用免疫組織化學方法觀察給予BAY 11-7082治療后的動物模型臨床表現(xiàn)和肌肉組織NLRP3與HMGB1的表達。3、采用免疫組織化學染色方法觀察6例PM、10例DM和10例肌活檢正常者(對照組)的肌肉組織NLRP3及HMGB1的表達。4、采用SWATH-MS技術檢測DM、PM、免疫壞死性肌病和假肥大型肌營養(yǎng)不良(均各為3例)患者的肌肉組織蛋白組學,并將其結(jié)果通過網(wǎng)站對差異蛋白進行分析比較。研究結(jié)果:1、EAM組小鼠的臨床表現(xiàn)明顯,血清CK含量升高,病理為典型肌炎改變,提示本模型為理想的EAM動物模型。2、與EAM組和對照組比較,EAM治療組臨床表現(xiàn)顯著好轉(zhuǎn)(1.73±0.1 vs 1.55±0.1,P0.01),四肢肌力顯著改善(11.0±0.7g/gvs8.1±0.2g/gvs13.4±0.7g/g,P0.01),肌肉組織NLRP3和HMGB1的mRNA及其蛋白表達均顯著下降(P0.01)。3、與對照組比較,PM患者肌肉組織NLRP3表達均為陽性,DM患者肌肉組織,NLRP3的表達陽性率為80%; PM患者肌肉組織HMGB1均表達陽性,DM患者肌肉組織HMGB1的表達陽性率為30%。4、與DMD組比較,PM組檢測出的差異蛋白有302種,DM組有484種;與NM組比較,PM組檢測出的差異蛋白有176種,DM組有261種。為尋找與炎性免疫性相關的差異蛋白,進一步分析發(fā)現(xiàn)DM組有6種,PM組有10種與炎癥相關。其中DM組的熱休克蛋白90-β和雙鏈糖蛋白與NLRP3炎性小體反應途徑相關。PM組中細胞凋亡相關的含有CARD區(qū)的斑點樣蛋白與NLRP3炎性小體反應途徑相關。研究結(jié)論:1、NLRP3及HMGB1共同參與特發(fā)性炎性肌病的發(fā)病過程。2、通過干預NLRP3及HMGB1的表達,可能有效治療效果。3、特發(fā)性炎性肌病可能存在與炎癥相關的生物標志蛋白。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R593.2

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