Purification of Growth Inhibitory Material from Bovine Brain

牛脑中生长抑制物质的纯化

• 批准号: 01571210
• 负责人: IDE Toshinori
• 金额: $ 1.09万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01571210/
• 关键词: Cell growth inhibition; Cell surface material; Bovine brain

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

本课题的目的是从生长停滞细胞的细胞表面纯化一种生长抑制物质。通过用蛋白水解酶或用磷脂酶处理从细胞表面释放生长抑制活性。恢复的活动被认为是不太有效的，但更可重复的脑细胞悬浮液处理磷脂酶C比蛋白酶。然而，由于磷脂酶C是昂贵的，我们选择蛋白酶用于进一步的实验。当用蛋白酶处理脑悬液时，在重复的实验中活性的恢复不同，并且即使经过广泛的改进试验也不能解决这个问题。当回收率较高时，进行进一步纯化。还检查了用于活性测定的细胞系。最后选用大鼠和人正常成纤维细胞系。这些细胞是第一次生长， ...更多信息 培养，然后用生长因子刺激生长，接着加入试验材料。通过将放射性胸苷掺入DNA中来监测生长抑制。用有机溶剂进一步提取从脑细胞表面回收的生长抑制活性，并将所得沉淀物冻干。将材料溶解在缓冲液中并应用于凝胶过滤柱上。在空隙体积（高分子量部分）和保留在柱中的部分中检测活性。回收空隙体积并浓缩。最后，回收吸附在糖亲和柱上的活性。该最终材料具有减少生长停滞的人和大鼠正常成纤维细胞中DNA合成诱导的活性。聚丙烯酰胺凝胶电泳和银染显示，该材料仍含有多种蛋白质。由于所获得的材料量太少，无法进一步纯化，因此应等待最终纯化。少

项目成果

中原 満: "Establishement of a SV40ーtransformed cell line from primary culture of rat dorsolateral prostetic epithelial cells." Experimental Cell Research. 190. 271-275 (1990)

Mitsuru Nakahara：“从大鼠背外侧假体上皮细胞原代培养物中建立 SV40 转化细胞系。”实验细胞研究。190. 271-275 (1990)

井出 利憲 (分担): "染色体と細胞周期" 丸善株式会社, 266(17)

Toshinori Ide（撰稿人）：“染色体和细胞周期”丸善株式会社，266（17）

高須賀剛: "A temperature-sensitive cell-cycle mutant of mammalian cells,tsJT16,is defective in a function operating soon after growth stimulation" Cell structure abd Function. 15. 39-45 (1990)

Tsuyoshi Takasuga：“哺乳动物细胞的温度敏感细胞周期突变体 tsJT16 在生长刺激后不久的功能上存在缺陷”《细胞结构 abd 功能》。

Yang Yu Tai, Jun Ninomiya-Tsuji, Kiyomi Furuoku, Naoko Ogawa, Sadahiko Ishibashi, Kazuko Shiroki, Kaoru Segawa, Nobuo Tsuchida, Masashi Shibuya, and Toshinori Ide: "Non-lethal GO-ts mutant tsJT60 becomes lethal at the non-permissive temperature after tran

Yang Yu Tai、Jun Ninomiya-Tsuji、Kiyomi Furuoku、Naoko Okawa、Sadahiko Ishibashi、Kazuko Shiroki、Kaoru Sekawa、Nobuo Tsuchida、Masashi Shibuya 和 Toshinori Ide：“非致命性 GO-ts 突变体 tsJT60 在非许可条件下变得致命

井出利憲: "哺乳類細胞の増殖調節" 細胞工学. 別冊5. 72-80 (1989)

Toshinori Ide：“哺乳动物细胞增殖的调节”《细胞工程》单独卷 5. 72-80 (1989)。