【作者】 赵忙所；

【导师】 耿同超；

【作者基本信息】 清华大学 ， 内科学， 2009， 硕士

【摘要】 视神经脊髓炎(neuromyelitis optica,NMO)是一种特发性中枢神经系统炎性脱髓鞘疾病,根据流行病学、临床特点、影像学、病理学及实验室检查,特别是血清NMO-IgG即抗水通道蛋白-4(aquaporin-4,AQP4)抗体等资料,国外大多数学者改变了NMO隶属于多发性硬化(multiple sclerosis,MS)的传统观点,认为它有别于MS,是一种全新的中枢神经系统自身免疫通道病。目前,尚未见中国内地NMO患者血清中存在抗AQP4抗体的报道,为了调查中国内地NMO患者血清中是否存在抗AQP4抗体,本研究建立了稳定表达AQP4蛋白的HEK293细胞株。从颞叶癫痫患者脑组织提取总RNA,使用RT-PCR扩增AQP4基因,构建AQP4-GFP融合表达重组体pEGFP-N1-AQP4,转染HEK293细胞,抗生素联合流式细胞仪筛选AQP4-GFP稳定表达细胞株。结果显示,其稳定表达率达80%~90%,RT-PCR和免疫荧光法证实该细胞株表达AQP4蛋白,激光共聚焦显微镜进一步确认AQP4-GFP融合蛋白主要表达于细胞膜上。在此基础上,我们收集了NMO、MS、神经系统其他疾病患者血清,并以健康正常人的血清作为阴性对照,利用已建立的稳定表达AQP4蛋白的HEK293细胞为“抗原”借助免疫荧光法检测了这些患者血清中的抗AQP4抗体。结果表明,中国内地NMO患者血清中也存在抗AQP4抗体,该方法对NMO诊断的灵敏度为88%,特异度为96.1%。为了建立适于临床推广的抗AQP4抗体简便有效的检测方法,我们初步探讨了以合成多肽为抗原的ELISA法检测NMO患者血清中的抗AQP4抗体,首次证明了AQP4蛋白细胞膜外的某些多肽段可作为ELISA检测抗原,用于检测患者血清中的抗AQP4抗体。总之,本研究证实了中国内地NMO患者血清中存在抗AQP4抗体,并初步建立了一个简便有效的NMO患者血清中抗AQP4抗体的检测方法,为我国NMO的诊疗提供了重要依据。更多还原

【Abstract】 Neuromyelitis optica (NMO) is an idiopathic inflammatory demyelinating disease in the central nervous system. Based on the epidemiological, clinical, radiological, pathological and laboratory findings, especially serum NMO-IgG which had been determined as anti-aquaporin-4 autoantibodies, recently most abroad researchers have changed the concept that NMO was a subset of multiple sclerosis(MS) and considered it as a new example of autoimmunological channelopathy in the central nervous system. So far, anti-AQP4 antibody in the serum of NMO patients in the mainland of China have not been reported.. In order to confirm if there is anti-AQP4 antibody in NMO patients’serum in Chinese mainland, HEK293 cell line stably expressing human AQP4 was set up. Total RNA was extrated from brain tissue of a patient diagnosed as temporal epliepsy. Human aquaporin-4 cDNA was amplied by RT-PCR and cloned into the plasmind pEGFP-N1. The recombinant plasmid was tansfected into HEK293, the stable cell line expressing AQP4-GFP fusion protein was screened by antibiotics and flow cytometry sorting. The result show that the rate of stable expression is 80~90%. The expression of AQP4 was detected by RT-PCR and immunofluorescence. The location of AQP4-GFP fusion protein was identified at the membrance under the laser confocal microscopy. Detect serum of NMO, MS, other neurological diseases and normal healthy people as negative control in the mainland of China with the cell line as“antigen”in immunofluorescence, it corfirmed the anti-AQP4 antibody in NMO serum in Chinese mainland, the sensitivity was 88%, specificity was 96.1%. Next,in order to set up a new simple assay to detect anti-AQP4 antibody in the serum of NMO patients which is accessible to clinic practice, we explored the ELISA with synthetic polypeptides as antigen for detecting AQP4 antibody in the serum of NMO patients and firstly testfied that some outmembrane polypeptides of AQP4 protein could be used as antigen in ELISA which can be used to detect anti-AQP4 antibody in the serum of NMO patients. In conclusions, the research identitied the existence of anti-AQP4 antibody in the serum of NMO patients in the mainland of China, and set up a new simple assay for detecting anti-AQP4 antibody in the serum of NMO patients, it would be helpful for diagnosis and therapy of NMO in China.更多还原