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

在鸡脑低温组织切片上培养新皮层细胞。细胞优先附着在成年大鼠中枢神经系统组织的灰质上。相比之下，在发育中的大鼠或成熟的青蛙脑组织中培养时，它们附着在大脑的任何部位。横断成年大鼠小脑上脚部纤维束，据报道导致小脑轴突再生，使附近的白质允许细胞附着。一个部分的灰质与另一个部分的白质重叠，将前者转化为不允许细胞附着的底物，证据表明，大鼠中枢神经系统对灰质的优先附着是由于白质中的抑制因子。这种抑制因子在青蛙和发育中的大鼠脑中似乎不存在。为了阐明抑制细胞附着的分子机制，我们试图从成年大鼠中枢神经系统中对该因子进行表征和纯化。在提取到氯仿/甲醇混合物后，将抑制活性溶于胆酸盐/脱氧胆酸盐溶液中。该因子需要重组为脂质体以表达其活性。经凝胶过滤（Superose 12），抑制活性在25-45 kD处达到峰值，该峰值与280 nm吸光度峰重合。用sds -聚丙烯酰胺电泳对含活性峰的组分进行分析时，检测到多个条带。薄层色谱还发现了脂质，可能是磷脂酰胆碱和-乙醇胺。因此，该因子可能是一种分子量相对较低的强疏水蛋白和脂质复合物。

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.