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.

我们之前的观察表明，大鼠质膜具有增强培养大鼠肝细胞中肝细胞生长因子诱导DNA合成的能力。本研究试图从大鼠肝细胞质膜中纯化这种致精因子并找到其作用位点。目前已获得如下结果： 1.纯化按照Hubbard等人的方法，对部分纯化的大鼠肝细胞质膜进行胶原酶消化，提取促有丝分裂活性，但得率太小，无法用于进一步纯化。电泳和柱制备。虽然用丙酮、正丁醇或CHAPS萃取可以将收率提高到原始活性的50%，但在用离子交换树脂制备柱后大部分活性丧失。用兔子制备的针对此类提取物的抗血清抑制了培养的大鼠肝细胞的DNA合成，但免疫印迹分析显示抗血清中存在许多条带。获得针对该因子的单克隆抗体的杂交瘤尚未成功。2.作用位点在培养的大鼠肝细胞中，质膜增加了腐胺的浓度，腐胺是肝细胞增殖所必需的多胺，同时完全消除了腐胺浓度的降低。添加 TGF β 诱导肝细胞进行 DNA 合成。当大鼠接受70%肝切除术时，肝脏中的促致精活性在6小时后逐渐降低，并在24小时时消失，但在31小时时恢复到正常水平。促有丝分裂因子可能在细胞周期的 G1 期起作用，与腐胺的产生有关，并在发挥其作用期间被失活或消耗。