【作者】 段枫；

【导师】 蒲传强；

【作者基本信息】 中国人民解放军军医进修学院 ， 神经病学， 2009， 博士

【摘要】 目的:1制作理想的实验性自身免疫性肌炎动物模型;2检测uPA、PAI-1、MMP-9、TIMP-2及肿瘤坏死因子-α在自身免疫性肌炎的肌肉组织中的表达,以探讨细胞外基质降解系统在多发性肌炎发病机制的重要作用。方法:1应用兔纯化的骨骼肌肌浆球蛋白（10mg/L）加完全弗氏佐剂4次皮下注射免疫豚鼠,同时联合腹腔注射百日咳杆菌原液(细菌数为4.0×10¹⁰个/只),制作实验性自身免疫性肌炎模型;通过临床观察观测动物临床表现并采用LennonLA评分法评估动物病变程度,应用酶法测定动物血CK,应用HE染色技术观察不同动物的肌肉组织病理变化。2采用逆转录PCR、实时定量PCR、免疫组织化学染色和Westernbloting方法检测正常组和EAM组豚鼠肌肉组织uPA、PAI-1、MMP-9、TIMP-2和TNF-alpha的基因mRNA及蛋白质表达。3采用美国Graphpad Prism 4软件分析,各项指标以均数±标准差（（?）±s）表示,对正常组和EAM组之间的计量资料进行t检验,P＜0.05表示差异有统计学意义。结果:1兔纯化的肌球蛋白加完全弗氏佐剂免疫豚鼠制作的动物模型的临床症状与病理改变与人类多发性肌炎类似;动物模型死亡率低,成功率高,病理改变典型。2 uPA在正常组动物肌肉组织间质仅有微量表达,但在EAM组动物肌肉组织间质呈强阳性表达,尤其是,uPA在炎性细胞的胞膜、坏变肌纤维的胞膜与胞浆上呈明显强阳性表达,肌纤维的胞膜表达更为显著。PAI-1在正常组动物肌肉组织间质也仅有微量表达,在EAM组动物肌纤维间质、炎性细胞周围、坏变肌纤维呈强阳性表达。EAM组动物肌肉组织uPA与PAI-1基因mRNA及蛋白质表达的水平比正常组显著增高（P＜0.001）,但其uPA/PAI-1基因mRNA表达的比例与正常组比较,则没有统计学差异（P＞0.05）。3 MMP-9在正常组动物肌肉组织间质仅有微量表达,但在EAM组动物间质出现广泛强阳性表达,其在炎性细胞周围、坏变肌纤维的胞膜和胞浆有广泛表达,尤其胞膜表达最显著。TIMP-2在正常组和EAM组动物肌肉组织的毛细血管、静脉管壁、动脉内皮细胞和神经鞘膜均有表达,且EAM组的表达比正常组更明显,不过,TIMP-2在正常组和EAM组动物肌肉组织的肌内衣、外衣没有表达;但是,TIMP-2在正常组动物肌纤维的胞膜和胞浆没有表达,而在EAM组动物肌纤维的胞膜和胞浆有表达,但表达的范围不广泛。EAM组动物肌肉组织中MMP-9和TIMP-2基因mRNA及蛋白表达水平显著高于正常组（P＜=0.001）,但EAM组MMP-9/TIMP-2基因mRNA表达的比例与正常组的表达有统计学差异（P＜0.001）。4免疫组织化学染色发现TNF-alpha在正常组肌肉组织没表达,但在EAM组动物肌肉组织的坏变肌纤维胞膜和胞浆及周围的炎性细胞均有表达。EAM组动物肌肉组织TNF-α基因mRNA及蛋白表达水平均显著高于正常组（P＜0.001）。结论1兔纯化的肌球蛋白加含卡介苗的完全弗氏佐剂可仿制出人类多发性肌炎的理想动物模型。2 UPA、PAI-1和TNF-α参与多发性肌炎的肌纤维坏变过程。3 MMP-9和TIMP-2在正常肌肉组织中的毛细血管、静脉管壁、动脉肉皮细胞和神经鞘膜表达,在多发性肌炎中,参与加重肌纤维的坏变过程。更多还原

【Abstract】 Objectives1 To investigate the methods of inducing ideal experimental autoimmune myositis（EAM）animal model that mimic human polymyositis.2 Evaluating the expression of the uPA、PAI-1、MMP-9、TIMP-2 and TNF-αin the EAM skeletal muscle in mRNA and protein level,to investigate the critical roles of extracellular matrix degregation system in the pathogenesis of PM.Methods1 EAM animal model was induced by pure rabbit myosin（10mg/l）mixed with CFA injected subcutaneously in guinea pigs,associated with bacillus pertussis(4.0×10¹⁰)injected intraperitoneally.We evaluated the model in clinical feature,the values of creatine kinase and pathological changes by LennonLA score,enzymic method and H&E staining technique.2 RT-PCR,Westen blot analysis and immunohistochemistry stain were used to evaluate the expression of u-PA,PAI-1,MMP-9,TIMP-2,TNF-αmRNA and protein in the skeletal muscle of the normal and EAM groups.3 The data were analyzed by Graphpad Prism 4 software.Comparison of data, presented as the mean±SD,was performed using t-test as indicated,p＜0.05 was considered significantly different.Results1 The clinical findings and pathologic changes in guinea pigs induced by pure Rabbit myosin mixed with CFA injected subcutaneously were similar to the human’s idiopathic inflammatory myopathy.The mortality of the models was low and achievement ratio was high.The changes in pathology were typical.2 The levels of the uPA and PAI-1 protein and mRNAs were increased significantly in the skeletal muscle of EAM guinea pigs compared with the levels in normal group.The ratio uPA to PAI-1 of transcriptional upregulation in the skeletal muscle was non-significant different between two groups,uPA and PAI-1 immunoreactivity were absent or weak in the skeletal muscle mesenchyma in normal group,but were significantly increased in EAM group. uPA and PAI-1 were strongly expressed in the degenerated,atrophic and necrotic fibers with or without inflammatory cells,and in the tissue space around the inflammatory cells in EAM muscle.3 The levels of MMP-9,TIMP-2 protein and mRNAs in the skeletal muscle of EAM guinea pigs were increased significantly compared with the levels observed in the normal controls.In EAM muscle,MMP-9 is strongly expressed in the degenerated,atrophic,necrotic fibers with or without inflammatory cells, and in the tissue space around the inflammatory cells.TIMP-2 also expressed in the diseased muscle that MMP-9 immune was positive,but the less immunoreactivity range was observed compared with MMP-9.TIMP-2 immunoreactivity was positive in the capillary,venous wall,arteriola endotheliocyte in the both groups skeletal muscle,but more intensive in EAM.4 The expression of TNF-αmRNA and protein was significantly up-regulated in the EAM muscle compared with the normal group.TNF-αimmunoreactivity were absent in the normal muscle,but were markedly up-regulated in the degenerated,necrotic,atrophy muscle fiber and around the inflammatory cells in the EAM muscle.Conclusion1 The EAM animal model induced by pure myosin mixed with CFA was similar to human’s polymyositis.It was considered an effective and ideal model of PM.2 uPA,PAI-1 and TNF-αwere all involved in the pathogenesis of PM and participated in the process of the degeneration,apoptosis and necrosis of the diseased muscle.3 MMP-9 and TIMP-2 immunoreactivity were positive in the capillary,venous wall,arteriola endotheliocyte and neurilemma in the normal group skeletal muscle,but more intensive in EAM.They may aggravate the pathological changes of the muscle in human PM.更多还原