Purification of Growth Inhibitory Material from Bovine Brain

本项目的目的是从生长阻滞细胞的细胞表面纯化一种生长抑制物质。将牛脑切碎，悬浮得到脑细胞悬浮液。通过蛋白水解酶或磷脂酶处理，生长抑制活性从细胞表面释放出来。研究发现，与蛋白酶相比，磷脂酶C处理脑细胞悬浮液的活性恢复效果较差，但可重复性更高。然而，由于磷脂酶C价格昂贵，我们选择了蛋白酶进行进一步的实验。用蛋白酶处理脑悬液后，在不同的重复实验中，活性的恢复情况有所不同，即使经过广泛的改善试验，这个问题也无法解决。当回收率较好时，进行进一步提纯。对用于活性测定的细胞系也进行了检测。最后选择大鼠正常成纤维细胞系和人正常成纤维细胞系。这些细胞首先生长，然后用生长因子刺激生长，然后添加测试材料。通过将放射性胸腺嘧啶掺入DNA来监测生长抑制。从脑细胞表面回收的生长抑制活性进一步用有机溶剂提取，所得沉淀物冻干。将物料溶解于缓冲液中，涂于凝胶过滤柱上。在空隙体积（高分子量分数）和柱中保留的分数中检测活性。空隙体积得到回收和浓缩。最后，恢复了吸附在糖亲和柱上的活性。这种最终材料具有降低生长受阻的人和大鼠正常成纤维细胞中DNA合成诱导的活性。然而，聚丙烯酰胺凝胶电泳和银染色显示，该材料仍含有多种蛋白质。由于获得的物质太少，无法进一步纯化，因此需要等待最终的纯化。少

The purpose of this project is to purify a growth inhibitory material from the cell surface of growth-arrested cells.Bovine brain was minced and suspended to get brain cell suspension. Growth-inhibiting activity was released from the cell surface by treatment with proteolytic enzymes or with phospholipase. Recovery of the activity was found to be less effective but more reproducible when brain cell suspension was treated with phospholipase C than with proteases. However, since phospholipase C was expensive we chose proteases for the further experiments. Recovery of the activity varied among repeated experiment when brain suspension was treated with protease, and this problem could not be solved even after an extensive trial for improvement. When we got good recovery, the further purification was carried out. The cell line used for the assay of the activity was also examined. Finally we chose rat normal fibroblast line and human normal fibroblast line. These cells were first growth arre … More sted and then growth-stimulated with growth factors followed by the addition of the test materials. Growth inhibition was monitored by the incorporation of radioactive thymidine into DNA. Growth inhibitory activity recovered from the brain cell surface was further extracted with organic solvents and the resulted precipitate was lyophilyzed. The material was dissolved in the buffer and applied on gel filtration column. The activity was detected both in the void volume (high molecular weight fraction) and in fractins retained in the column. The void volume was recovered and concentrated. Finally, the activity that adsorbed on the affinity column for a sugar was recovered. This final material had an activity to reduce an induction of DNA synthesis in growth-arrested human and rat normal fibroblasts. However, this material still contained several kinds of proteins as revealed by polyacrylamidgel electrophoresis and silver staining. Since obtained materials were too small amount to purify further, the final purification should be awaited. Less