Myelination in coculture of established neuronal and Schwann cell lines
Myelination in coculture of established neuronal and Schwann cell lines
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DOI：
10.1007/s00418-012-0934-3
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发表时间：
2012-06-01
影响因子：
2.3
通讯作者：
Watabe, Kazuhiko
中科院分区：
文献类型：
--
作者：
Sango, Kazunori;Kawakami, Emiko;Watabe, Kazuhiko
与神经细胞和雪旺细胞系建立稳定的共培养体系一直被认为是困难的，可能是因为它们具有高的增殖活性和与原代培养细胞的表型差异。本研究旨在探讨神经沟来源的嗜铬细胞瘤细胞系PC12与永生化的成年大鼠雪旺细胞系IFRS1共培养条件下髓鞘形成的方法。共培养前，将PC12细胞以低密度(3×10(2)/cm(2))接种于含氮、抗坏血酸(50mU g/ml)和神经生长因子(NGF)(50 ng/ml)的无血清培养液中培养1周。暴露在这种富含NGF和最少营养的环境中，可以加速PC12细胞的分化和轴突延伸，但不会促进其增殖。在NGF刺激的PC12细胞中加入IFRS1细胞后，PC12细胞与IFRS1细胞的密度比由1：50调整为1：100。然后将共培养的细胞置于添加B27、抗坏血酸(50mU g/ml)、神经生长因子(10 ng/ml)和重组可溶性NeuRegin-1 III型(25 ng/ml)的无血清培养液中。共培养第28天，光镜和电子显微镜观察髓鞘形成情况。稳定的PC12-IFRS1共培养体系不存在原代培养产生的技术和伦理问题，是研究周围神经变性和再生的有价值的工具。
Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 x 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 mu g/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 mu g/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.