McLoon L K, McLoon S C
Department of Ophthalmology, University of Minnesota, Minneapolis 55455.
Brain Res. 1988 Mar 1;467(1):61-8. doi: 10.1016/0165-3806(88)90067-3.
We have examined the ability of Schwann cell-conditioned medium (SCCM) to promote neurite growth from embryonic retina and tectum in explant culture. Both retinal and tectal explants adhered to polylysine substrates, but neurite outgrowth from the explants was minimal when grown in the presence of non-conditioned serum-free defined medium. The addition of SCCM resulted in a significant extension of neurites from both types of explants. The neurite outgrowth from the retinal explants was mainly radial and appeared to be in fascicles of small diameter. Neurite outgrowth from the tectal explants stimulated by the SCCM tended to be in larger fascicles and in a more convoluted pattern than that seen from the retinal explants. After serial passaging of the SCCM to remove any substrate-adsorbable factors all neurite-promoting activity was lost. Neurite growth from 100% of the explants was seen on the tissue culture plates that had been preincubated with the SCCM. SCCM was analyzed by immunoblot to determine whether it contained laminin, a potent promoter of neurite outgrowth. Samples of media were electrophoresed on a sodium dodecyl sulphate (SDS)-polyacrylamide gel alongside purified laminin, transferred to nitrocellulose and stained with an antibody to laminin. Purified laminin ran in two bands at 400 kDa and 200 kDa. The conditioned media had a laminin positive band at 200 kDa. Antibody to laminin, when added to the conditioned media, resulted in a loss of the neurite promoting activity of the SCCM. These results suggest that the conditioned media contained laminin or a laminin-like molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究了雪旺细胞条件培养基（SCCM）在外植体培养中促进胚胎视网膜和顶盖神经突生长的能力。视网膜和顶盖外植体均能附着于聚赖氨酸底物上，但在未条件化的无血清限定培养基中生长时，外植体的神经突生长极少。添加SCCM后，两种外植体的神经突均显著延长。视网膜外植体的神经突生长主要呈放射状，似乎以小直径束状形式存在。SCCM刺激顶盖外植体产生的神经突生长往往形成更大的束状，且比视网膜外植体的更为曲折。对SCCM进行连续传代以去除任何可吸附于底物的因子后，所有促进神经突生长的活性均丧失。在预先用SCCM孵育过的组织培养板上，100%的外植体均出现神经突生长。通过免疫印迹分析SCCM，以确定其是否含有层粘连蛋白，层粘连蛋白是一种有效的神经突生长促进剂。将培养基样品与纯化的层粘连蛋白一起在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上进行电泳，转移至硝酸纤维素膜上，并用层粘连蛋白抗体染色。纯化的层粘连蛋白在400 kDa和200 kDa处出现两条带。条件培养基在200 kDa处有一条层粘连蛋白阳性带。将层粘连蛋白抗体添加到条件培养基中会导致SCCM促进神经突生长的活性丧失。这些结果表明，条件培养基中含有层粘连蛋白或类层粘连蛋白分子。（摘要截短至250字）
