人正常神经组织许旺细胞的体外培养

Human Schwann cells from normal nervous tissue cultured in vitro

背景:周围神经组织工程所需要的许旺细胞数量巨大。在以往的研究中,由于正常人神经组织的缺乏,常选用大鼠、兔等动物神经组织分离许旺细胞进行培养,而作为异种细胞在临床的应用相对受限。目的:体外培养人正常周围神经的许旺细胞,寻找一种最佳的获取、培养、纯化的方法。方法:切取脑瘫患者周围神经缩窄术中的正常周围神经,以消化培养法为主,结合差速贴壁法纯化培养许旺细胞。将神经组织剪成碎块,接种于含胎牛血清、胶原酶、Dispase酶的培养液中消化培养,离心,将组织块加入培养基中,吹打成单细胞悬液,再移入有多聚赖氨酸的DMEM培养皿中,加入碱性成纤维细胞生长因子培养,当贴壁细胞覆盖培养皿达85%～90%即可开始传代培养。锥虫蓝染色对培养后的第2,3,4,5,6,7,8,9,10天不同时间许旺细胞进行计数,并经S-100蛋白免疫组织化学染色鉴定计算细胞纯度。结果与结论:4d后镜下可见基本为纯净的许旺细胞,密度在0.5×108L-1以上。传3代后,许旺细胞计数可达到9×108L-1以上;许旺细胞纯度达85%以上。结果表明,以消化培养法结合差速贴壁方法培养许旺细胞所需时间短,可获得较高纯度的人许旺细胞,能够为进一步的神经组织工程提供细胞来源。

BACKGROUND： Peripheral nerve tissue engineering needs a large number of Schwann cells. In previous studies, lack of normal human nervous tissue, so animal （rat, rabbit, et al） nervous tissues are commonly used to isolate Schwann cells, but as xeno-cells it is limited in clinical application. OBJECTIVE： To investigate an effective technique for isolation, cultivation and purification of human Schwann cells of normal peripheral nerves cultured in vitro. METHODS： Normal peripheral nerves were obtained from the surgery of cerebral palsy patients. Schwann cells were cultured with enzymatic digestion culture method and differential attachment method. Tissues were cut into pieces and incubated in medium supplemented with fetal bovine serum, collagenase and Dispase enzyme, centrifuged. Tissue blocks were placed in the medium, triturated into monoplast suspension, and then moved into a DMEM Petri dish containing polylysine, supplemented with basic fibroblast growth factor. When adherent cells were confluent about 85%-90%, cells could subculture. Schwann cells were counted by Trypan Blue coloring method at 2, 3, 4, 5, 6, 7, 8, 9, 10. The purity of Schwann cells was identified through S-100 protein immunohistochemistry staining. RESULTS AND CONCLUSION： More than 0.5×108/L Schwann cells were detected after four days under a microscope. Following the third passage, the number of Schwann cells was over 9×108/L. The purity of Schwann cell population was up to 85%. Results suggested that plenty and purified human Schwann cells could be obtained by enzymatic digestion culture and differential attachment methods in a short time, which can be used for the source of peripheral nerve tissue engineering.