中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (6): 903-908.doi: 10.12307/2023.262

• 干细胞外泌体 Stem cell exosomes • 上一篇    下一篇

脑性瘫痪儿童脑脊液内外泌体的蛋白质组学分析

张厚君,邓博文,蒋昇源,赵  毅,任敬佩,徐  林,穆晓红   

  1. 北京中医药大学东直门医院,北京市   100105
  • 收稿日期:2022-02-23 接受日期:2022-05-11 出版日期:2023-02-28 发布日期:2022-08-11
  • 通讯作者: 穆晓红,博士,主任医师,北京中医药大学东直门医院,北京市 100105
  • 作者简介:张厚君,男,1989年生,山东省临沂市人,汉族,北京中医药大学在读博士,主要从事脊柱疾病和脑瘫外科治疗。
  • 基金资助:
    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

Proteomic analysis of cerebrospinal fluid exosomes derived from cerebral palsy children

Zhang Houjun, Deng Bowen, Jiang Shengyuan, Zhao Yi, Ren Jingpei, Xu Lin, Mu Xiaohong   

  1. Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100105, China
  • Received:2022-02-23 Accepted:2022-05-11 Online:2023-02-28 Published:2022-08-11
  • Contact: Mu Xiaohong, MD, Chief physician, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100105, China
  • About author:Zhang Houjun, Doctoral candidate, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100105, China
  • Supported by:
    Frontier Project of Beijing Natural Science Foundation-Haidian Original Innovation Joint Fund, No. 19L2059 (to MXH)

摘要:

文题释义:
脑瘫:脑瘫全称为脑性瘫痪,指出生前至出生后早期,脑发育未成熟阶段受到永久、非进行性损伤,形成以运动障碍和姿势异常为主的综合征。
外泌体:是指包含了复杂 RNA 和蛋白质的小膜泡(30-150 nm),现今,其特指直径在40-100 nm的盘状囊泡。

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。

https://orcid.org/0000-0002-6317-6981 (张厚君) 

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 脑瘫, 脑脊液, 外泌体, 蛋白质组学分析

Abstract: 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 cerebrospinal fluid of children with cerebral palsy, and exosome proteins may be related to the molecular pathological mechanism of cerebral palsy.

Key words: cerebral palsy, cerebrospinal fluid, exosome, proteomic analysis

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