中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (9): 1677-1685.doi: 10.4103/1673-5374.306093

• 原著:脑损伤修复保护与再生 •    下一篇

原代小胶质细胞和星形胶质细胞移植对缺血性脑卒中神经干细胞的作用:体内外动物实验

  

  • 出版日期:2021-09-15 发布日期:2021-02-05
  • 基金资助:

    中国国家自然科学基金项目(813712138107098730971531),中国科学技术部项目(2010CB9456002010CB945601

Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke

Sheng-Jun Wen, Xi-Min Zheng, Li-Fen Liu, Na-Na Li, Hai-An Mao, Liang Huang, Qiong-Lan Yuan*#br#   

  1. Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China
  • Online:2021-09-15 Published:2021-02-05
  • Contact: Qiong-Lan Yuan, yqiongl@tongji.edu.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 81371213, 81070987, 30971531, and grants from the Ministry of Science and Technology of China, Nos. 2010CB945600, 2010CB945601 (all to QLY).

摘要:

神经干细胞移植对脑卒中神经元保护作用已在动物实验得以证实。模型显示出好的治疗效果,但在移植后的神经干细胞低存活率和低神经元的分化率限制了其临床应用。神经干细胞所处的胶质微环境能调节神经干细胞的存活、增殖与分化。但是脑卒中后激活的胶质细胞对神经干细胞的影响并不清楚。实验拟通过体内外方式探索脑卒中激活的胶质细胞对神经干细胞的影响,以及神经干细胞与胶质细胞的联合移植是否可以对脑卒中产生更好的作用。(1)首先将原代培养的星形胶质细胞、小胶质细胞或混合神经胶质细胞暴露于Transwell共培养体系谷氨酸或过氧化氢中接种于上层,原代神经干细胞接种于下层,共培养7d。发现小胶质细胞有助于神经球的形成,且对神经球的凋亡没有影响;而星形胶质细胞和混合神经胶质细胞虽有助于神经分化,并抑制细胞凋亡。这一结果不受谷氨酸或过氧化氢预处理的影响。除过氧化氢预处理的星形胶质细胞外,其他预处理均不影响小胶质细胞和星形胶质细胞诱导神经元分化;(2)以大脑中动脉闭塞建立缺血性脑卒中大鼠模型,3d后右侧脑室注射5 × 105个与小胶质细胞或星形胶质细胞共培养的神经干细胞。结果显示神经干细胞与星形胶质细胞联合移植干预缺血性脑卒中大鼠后4d,其神经功能优于单独神经干细胞移植组;且与神经干细胞移植组相比,神经干细胞与星形胶质细胞或小胶质细胞联合移植显著缩小了脑梗死体积;(3)表明小胶质细胞和星形胶质细胞对神经干细胞的不同作用,且神经干细胞与星形胶质细胞共移植有利于缺血性脑卒中大鼠神经功能的恢复。实验于2010年经同济大学医学院动物伦理委员会批准,批准号2010-TJAA08220401。

https://orcid.org/0000-0001-6165-2107 (Qiong-Lan Yuan)

关键词:

神经干细胞, 神经元, 小胶质细胞, 星形胶质细胞, 脑卒中, 谷氨酸, 过氧化氢, 修复, 神经发生

Abstract: Transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models; however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H2O2 and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H2O2. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 105 neural stem cells  with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem cell-treated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China (approval No. 2010-TJAA08220401) in 2010. 

Key words: astrocytes, glutamate, microglia, neural stem cells, neurogenesis, neurons, peroxide, repair, stroke