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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.

我们以前的观察表明，大鼠质膜有能力提高诱导的DNA合成的肝细胞生长因子在培养的大鼠肝细胞。本研究试图从大鼠肝细胞质膜中分离纯化该共促因子，并寻找其作用部位。目前已获得以下结果：1.纯化通过胶原酶消化部分纯化的大鼠肝细胞的质膜来提取共有丝分裂活性，根据Hubbard等人的方法，但产量太小，不能用于通过电泳和柱制备的进一步纯化。虽然通过用丙酮、正丁醇或CHAPS萃取，产率增加到原始活性的50%，但用离子交换树脂柱制备后，大部分活性丧失。用兔制备的抗血清抑制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有许多条带。2.作用部位：细胞质膜能增加肝细胞增殖所必需的多胺腐胺的浓度，同时能完全消除TGF β诱导的肝细胞DNA合成的下降。当大鼠行70%肝切除术时，肝内共生因子活性在6小时后逐渐降低，24小时消失，31小时恢复正常，共生因子可能作用于细胞周期的G1期，与腐胺的产生有关，在发挥作用过程中被灭活或消耗。