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

我们之前的观察表明，在培养的大鼠肝细胞中，大鼠质膜具有增强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该致comitogenic factor，并寻找其作用位点。到目前为止，得到了以下结果：根据Hubbard等人的方法部分纯化的大鼠肝细胞质膜，通过胶原酶消化提取其产comitogenic activity，但产量太小，无法用于进一步的电泳和柱制备纯化。虽然丙酮、正丁醇或CHAPS萃取使产率提高到原活性的50%，但用离子交换树脂制备柱后，大部分活性丧失。兔提取的抗血清对培养的大鼠肝细胞的DNA合成有抑制作用，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。作用部位(s)质膜增加了培养大鼠肝细胞中肝细胞增殖所必需的多胺腐胺的浓度，同时完全消除了TGF β对肝细胞DNA合成的抑制作用。当大鼠肝切除70%后，6小时后肝脏产粪活性逐渐下降，24小时后消失，31小时后恢复正常。产粪因子可能在与腐胺产生相关的细胞周期g1期起作用，并在发挥作用时被灭活或消耗。

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.