Purification of hepatocyte comitogenic factor in plasma membrane and its action site (s) in hepatocyte proliferation.

质膜中肝细胞有丝分裂因子的纯化及其在肝细胞增殖中的作用位点。

基本信息

批准号：
04454240
负责人：
FUJIWARA Kenji
金额：
$ 4.42万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454240/
关键词：
Hepatocyte plasma membrane Hepatocyte growth factor liver regeneration Putrescine TGFbeta 劇症肝炎

项目摘要

Our previous observations show that rat plasma membrane has the ability to enhance induction of DNA synthesis by hepatocyte growth factors in cultured rat hepatocytes. The present study was attempted to purify this comitogenic factor from plasma membrane of rat hepatocytes and find its action sites. The following results have been so far obtained.1.PurificationThe comitogenic activity was extracted by collagenase digestion of the plasma membrane of rat hepatocytes partially purified according to the method of Hubbard et al., but the yield was too small to use for further purification by electrophoresis and column preparations. Although the yield was increased to 50% of the original activity by extraction with acetone, n-butanol or CHAPS,most of the activity was lost after column preparations with ion exchange resins. Antiserum against such extracts made using rabbits inhibited DNA synthesis by cultured rat hepatocytes, but immunoblot analysis revealed a lot of bands in the antiserum. To obtain hybridoma for a monoclonal antibody against the factor has not yet been successful.2.Action site(s)The plasma membrane increased concentrations of putrescine, an essential polyamine for hepatocyte proliferation, in cultured rat hapatocytes, while it abolished completely the decrease in DNA synthesis by the hepatocytes induced by ddition of TGF beta. When rats underwent 70% hepatectomy, the comitogenic activity in the liver was gradually decreased after 6 hr and disappeared at 24 hr, but recovered to normal levels at 31 hr. The comitogenic factor might act at the G1-phase of the cell cycle in association with putrescine production, and be inactivated or consumed during exerting its action.

我们先前的观察结果表明，大鼠质膜具有在培养的大鼠肝细胞中通过肝细胞生长因子增强DNA合成的能力。本研究试图从大鼠肝细胞的质膜中净化这种连质因子，并找到其作用位点。 1. purification通过胶原酶消化提取了脑源性活性。大鼠肝细胞的质膜的胶原酶消化，根据Hubbard等人的方法部分纯化了肝细胞的质膜，但是产量太小，无法通过电泳和柱制剂进一步纯化，无法用来进一步纯化。尽管通过用丙酮，N-丁醇或CHAP提取型的产量增加到原始活性的50％，但在用离子交换树脂制备后，大部分活动都是损失的。使用兔子制成的这种提取物的抗血清通过培养的大鼠肝细胞抑制DNA的合成，但免疫印迹分析显示抗血清中有很多带。为了获得针对该因子的单克隆抗体的杂交瘤。2。质膜增加plasmrane plassin的浓度增加，腐霉菌是肝细胞增殖的必不可少的多胺，在培养的大鼠hapaTopytes中，它完全消除了DNA合成的降低，而DNA合成中的降低是通过hepatocteS in Incpatience的降低。当大鼠接受70％的肝切除术时，肝脏中的闭合活性在6小时后逐渐降低并在24小时消失，但在31小时时恢复到正常水平。连骨因子可能在细胞周期的G1期起作用，与腐败产生相关，并在发挥作用时被灭活或消耗。