摘要
背景:脑性瘫痪(简称脑瘫)是一种广泛的神经损伤性疾病,其病因在很大程度上仍不明确,用于该病分类或监测的分子指标也尚未见报道。目的:分离培养儿童脑瘫患者脑脊液来源的外泌体,使用无标记定量质谱蛋白质组学方法分析其外泌体的蛋白质组表达谱特征,为儿童脑瘫群体的分型、诊断和预后评估等提供分子生物学依据。方法:选择在北京中医药大学东直门医院进行选择性脊神经后根切断术的脑瘫患者,其中痉挛型脑瘫患者4例,混合型脑瘫患者3例,每位患者抽取3 mL脑脊液,通过超速离心法分离儿童脑瘫患者脑脊液中的外泌体,并使用纳米颗粒跟踪分析、透射电镜和蛋白质质谱分析对外泌体进行表征。结果与结论:①透射电镜观察显示,儿童脑瘫患者脑脊液内存在外泌体形态的囊泡。Western blot检测到外泌体标志蛋白Syntenin-1和Flotillin-1的表达;②纳米颗粒跟踪分析显示囊泡的大小和浓度符合外泌体的异质性特征。对痉挛性脑瘫、混合性脑瘫的脑脊液外泌体进行蛋白质谱分析,共鉴定出551个蛋白;③生物信息学分析显示,鉴定出的脑脊液外泌体蛋白与负责外泌体功能和神经发育过程的分子信号机制相关,这些蛋白主要富集于额叶皮质、运动皮质等脑部区域,与脑瘫病理密切相关。研究结果提示儿童脑瘫患者脑脊液内存在外泌体,外泌体蛋白可能与脑瘫分子病理机制相关。
BACKGROUND:Cerebral palsy is a wide range of nerve injury diseases.Its etiology is still unclear to a great extent,and the molecular indicators used for classification or monitoring of the disease have not been reported.OBJECTIVE:To isolate and culture cerebrospinal fluid derived exosomes from children with cerebral palsy,and analyze the proteomic expression profile characteristics of exosomes by label-free quantitative mass spectrometry proteomics,so as to provide molecular biological basis for typing,diagnosis and prognosis evaluation of children with cerebral palsy.METHODS:Among cerebral palsy patients undergoing selective posterior rhizotomy at Dongzhimen Hospital of Beijing University of Chinese Medicine,there were four patients with spastic cerebral palsy and three patients with mixed cerebral palsy.3 mL cerebrospinal fluid was extracted from each patient.The exosomes in the cerebrospinal fluid of children with cerebral palsy were separated by ultracentrifugation,and characterized by nanoparticle tracking analysis,transmission electron microscope and protein mass spectrometry.RESULTS AND CONCLUSION:(1)Transmission electron microscopy showed that there were secretory vesicles in cerebrospinal fluid of children with cerebral palsy.Western blot assay detected the expression of secrete marker proteins Syntenin-1 and Flotillin-1.(2)Nanoparticle tracking analysis showed that the size and concentration of vesicles were consistent with the heterogeneity of exosomes.The cerebrospinal fluid exosomes of spastic cerebral palsy and mixed cerebral palsy were analyzed by protein mass spectrometry.A total of 551 proteins were identified.(3)Bioinformatics analysis showed that the identified cerebrospinal fluid exosome proteins were related to the molecular signal mechanism of exosome function and neural development.These proteins were mainly concentrated in brain regions such as frontal cortex and motor cortex,which were closely related to the pathology of cerebral palsy.It is concluded that there are exosomes in cerebrospi
作者
张厚君
邓博文
蒋昇源
赵毅
任敬佩
徐林
穆晓红
Zhang Houjun;Deng Bowen;Jiang Shengyuan;Zhao Yi;Ren Jingpei;Xu Lin;Mu Xiaohong(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100105,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第6期903-908,共6页
Chinese Journal of Tissue Engineering Research
基金
北京市自然科学基金-海淀原始创新联合基金前沿项目(19L2059),项目负责人:穆晓红。
关键词
脑瘫
脑脊液
外泌体
蛋白质组学分析
cerebral palsy
cerebrospinal fluid
exosome
proteomic analysis
