中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (2): 289-297.doi: 10.4103/1673-5374.226398

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

硫酸软骨素蛋白聚糖对体外培养神经元黏附、铺展和神经突生长的抑制

  

  • 收稿日期:2017-12-20 出版日期:2018-02-15 发布日期:2018-02-15

Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture

Jingyu Jin1, Sharada Tilve2, Zhonghai Huang1, Libing Zhou1, Herbert M. Geller2, Panpan Yu1   

  1. 1 Guangdong-Hongkong-Macau Institute of CNS Regeneration; Ministry of Education Joint International Research Laboratory of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China
    2 Laboratory of Developmental Neurobiology, Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
  • Received:2017-12-20 Online:2018-02-15 Published:2018-02-15
  • Contact: Panpan Yu, Ph.D.,yupanpan21@jnu.edu.cn.
  • Supported by:

    This work was supported by the National Natural Science Foundation of China, No. 81601066; the Natural Science Foundation of Guangdong Province of China, No. 2017A030313103 and 2016A030313096; a grant from the Program of Introducing Talents of Discipline to Universities, No. B14036; the Fundamental Research Funds for the Central Universities, No. 21616340; and the Division of Intramural Research of the National Heart, Lung, and Blood Institute of National Institutes of Health.

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摘要：

长期以来，硫酸软骨素蛋白聚糖（CSPGs）作为中枢神经系统细胞外基质（ECM）的一种主要成分，对中枢神经系统损伤后轴突生长期有抑制作用。虽然许多体外研究证实，硫酸软骨素蛋白聚糖可抑制多种神经元神经突生长，但其原因尚有待进一步研究。为此，实验对小脑颗粒神经元（CGN）进行培养，评估不同浓度的固定化和可溶性硫酸软骨素蛋白聚糖对神经元黏附、铺展和轴突生长行为的影响。通过免疫组化染色观察到，当硫酸软骨素蛋白聚糖浓度升高时，神经突长度减小，细胞密度降低，并伴随着细胞聚集体形成的增加。可溶性硫酸软骨素蛋白聚糖也显示出对神经突生长的抑制作用，但是其在诱导细胞聚集体形成方面，需要比涂覆的硫酸软骨素蛋白聚糖更高的浓度。实验还发现，由于硫酸软骨素蛋白聚糖对板状伪足扩张的抑制作用，神经元生长锥尺寸明显缩小，神经细胞的铺展也受到限制。干扰反射显微镜（IRM）进一步证实了硫酸软骨素蛋白聚糖对神经元黏附的影响，直接证明了小脑颗粒神经元和大脑皮质神经元更松散地黏附于硫酸软骨素蛋白聚糖底物。总之，这些数据表明硫酸软骨素蛋白聚糖对神经元黏附、铺展、神经突生长均有明显的抑制作用，且该作用呈剂量依赖性。

orcid:0000-0002-4479-1479(Panpan Yu)

关键词: 硫酸软骨素蛋白聚糖, 细胞粘附, 轴突生长, 干涉反射显微镜, 神经再生

Abstract:

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Key words: chondroitin sulfate proteoglycans, cell adhesion, neurite growth, interference reflection microscopy, neural regeneration