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 (11): 1893-1899.doi: 10.4103/1673-5374.239438

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

商品化髓磷脂相关蛋白MAG-Fc适用于抑制神经突起生长的实验研究

  

  • 收稿日期:2018-07-23 出版日期:2018-11-15 发布日期:2018-11-15
  • 基金资助:

    中国国家自然科学基金项目（81171178）

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Fu Liu1, Mei-Ling Gao1, 2, Juan Bai1, Ya-Fang Wang1, Xia-Qing Li1   

  1. 1 Department of Pathophysiology, Shanxi Medical University, Taiyuan, Shanxi Province, China
    2 Department of Vascular Surgery, People’s Hospital of Gansu Province, Lanzhou, Gansu Province, China
  • Received:2018-07-23 Online:2018-11-15 Published:2018-11-15
  • Contact: Xia-Qing Li, PhD,xqli2013@sxmu.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81171178.

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摘要：

髓磷脂相关糖蛋白可抑制神经细胞中突起的生长。由于内源性髓磷脂相关糖蛋白提取需要较为复杂的操作，出于经济及人力考虑，作者试图确定商品化的MAG-Fc是否可替代内源性髓磷脂相关糖蛋白用于研究。(1)通过免疫荧光检测髓磷脂相关糖蛋白的2种特异性抗体NgR和PirB在neuro-2a细胞的阳性反应，以确定MAG-Fc是否可以被内吞，此外评估不同剂量磷脂相关糖蛋白干预的neuro-2a细胞的神经突增生。(2)以免疫荧光染色检测髓磷脂相关糖蛋白和III型微管蛋白的阳性反应进行验证。经髓磷脂相关糖蛋白干预后，通过ELISA测定RhoA的活性，并通过Western blot评估ROCK的磷酸化。(3)结果表明，neuro-2a细可表达NgR和PirB，且MAG-Fc可通过其与NgR和PirB的结合而被内吞，进一步激活细胞内信号传导途径，包括增加RhoA活性和磷酸化ROCK，最终抑制神经突生长。(4)数据验证了MAG-Fc可通过激活RhoA信号通路如内源性髓磷脂相关糖蛋白来抑制神经轴突的生长，而且还清楚地证明了商品化髓磷脂相关蛋白MAG-Fc适用于抑制神经突起生长的相关实验研究。

orcid:0000-0003-4563-8801(Xia-Qing Li)

 

关键词: 髓鞘生长抑制剂, 髓鞘相关糖蛋白, MAG-Fc, 细胞培养, 髓磷脂相关糖蛋白受体, neuro-2a细胞, RhoA/ROCK信号通路, 神经突生长

Abstract:

Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Key words: nerve regeneration, myelin growth inhibitors, myelin-associated glycoprotein, MAG-Fc, cell culture, receptors for myelin-associated glycoprotein, neuro-2a cell line, RhoA/ROCK signaling pathways, neurite outgrowth, neural regeneration