乳鼠坐骨神经植块法的雪旺氏细胞培养

目的:改善并建立一种新的大鼠雪旺氏细胞(SCs)的培养方法,为研究外周神经损伤修复模型及其它外周神经相关实验提供高纯度、多数量的SCs。方法:麻醉后显微镜下解剖并分离新生3天内SD大鼠的坐骨神经,采取植块培养的方法,显微镜下尽量剥除坐骨神经纤维外膜,并梳理松解坐骨神经的神经纤维束。梳理后剪碎坐骨神经,每小块种植于培养皿中,使用纯血清培养4小时,再加入正常的DMEM/F12培养基,消化培养2-3代。最后用S-100及GFAP免疫荧光染色进行纯度鉴定。结果:本实验在总结前人实验的基础上,联合创新采用坐骨神经外膜剥除、神经内膜梳理、纯血清培养以及胰酶差速消化等方法,短时间内获得SCs的纯度可达99%以上,可用于进一步对雪旺氏细胞的功能进行研究。结论:这种选用乳鼠坐骨神经植块、血清培养的方法简单易操作,无需额外的生长因子及抑制因子,可在短期内获得大量高纯度的SCs。

Culture of Schwann Cells from Sciatic Nerve of Newborn Rat in Vitro

ABSTRACT Objective: To improve and establish a new culture method of Schwann cells (SCs), and to provide a high purity and majority of Schwann cells for the study of peripheral nerve injury and other relative researches. Methods: Newborn 3 days SD rat sciatic nerve were dissected and isolated under anaesthesia, taking the method of block culture to microscopic stripe of epineurium and comb endoneurium. After cutting, the explants were cultured in pure serum for 4 hours, and then added normal DMEN/F12 medium to culture. After 2-3 generations of digest and culture, finally, S-100 and GFAP immunohistochemistry were used to determine the purity. Results: The purity of Schwann cells was above 99 % after sciatic nerve explant and serum culture. Conclusion: This method is simple and easy to operate, and it can obtain a large number of Schwann cells in a short time without additional growth factors and inhibitors.