Inhibition of Cell Attachment and Neurite Outgrowth by the Factors in Abult CNS White Matter : a Studbstrata.

将鸡新皮质细胞在脑的低温恒温器组织切片上培养。细胞优先附着于成年大鼠中枢神经系统组织的灰质。相反，在发育中的大鼠或青蛙脑组织上培养时，它们可以附着在大脑的任何部位。切断成年大鼠小脑上级脚交叉处的纤维束，据报道可引起离小脑轴突的再生，使附近的白色允许细胞附着。将一个切片的灰质叠加到另一个切片的白色物质上，将前者转化为细胞附着的非允许基质，证据表明，细胞优先附着到成年大鼠CNS的灰质是由于位于白色物质中的抑制因子。这种抑制因子在蛙脑和发育中的大鼠脑中似乎是不存在的，为了阐明抑制细胞粘附的分子机制，我们试图从成年大鼠中枢神经系统中表征和纯化该因子。将抑制活性溶解到胆酸盐/脱氧胆酸盐溶液中，然后萃取到氯仿/甲醇的混合物中。该因子需要重构到脂质体中以表达其活性。通过凝胶过滤（Superose 12），抑制活性在25-45 kD处达到峰值，并且该活性峰与280 nm吸光度的峰一致。当用SDS-聚丙烯酰胺电泳分析包括峰活性的级分时，检测到几条带。薄层层析也显示脂质，可能是磷脂酰胆碱和乙醇胺.因此，该因子可能是强疏水性蛋白质和相对低分子量的脂质的复合物。

Chick neocortical cells were cultured on cryostat tissue sections of the brain. Cells preferentially attached to the gray matter of adult rat CNS tissues. In contrast, they attached to any part of the brain when cultured on developing rat or marure frog brain tissues. Transection of fiber bundles at the superior cerebellar peduncle decussation of adult rat, which reportedly causes regeneration of cerebellofugal axons, made nearby white matter permissive to cell attachment. Superimposition of the gray matter of one section onto the white matter of another converted the former into a nonpermissive substrate for cell attachment, evidence suggesting that preferential cell attachment to the gray matter of abult rat CNS is due to inhibitory factor (s) localized in the white matter. This inhibitory factor appears to be absent in frog brain and developing rat brain.To clarify the molecular mechanisms subserving the inhibition of cell attachment, we attempted to characterize and purify the factor from adult rat CNS. The inhibitory activity was solubilized into cholate/deoxycholate solution following extraction into a mixture of chroloform/methanol. This factor required reconstitution into liposomes for expression of its activity. By gel filtration (Superose 12), the inhibitory activity peaked at 25-45 kD and this peak of activity coincided with a peak of 280 nm absorbance. When the fractions including the peak activity were analyzed by SDS-polyacrylamide electrophoresis, several bands were detected. Thin layer chromatography also revealed lipids, possibiy phosphatidyl -choline and -ethanolamine. Hence the factor might be a complex of strongly-hydrophobic protein (s) and lipid (s) with relatively low molecular weight.