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

我们先前的观察表明，大鼠肝细胞质膜具有增强肝细胞生长因子诱导大鼠肝细胞DNA合成的能力。本研究试图从大鼠肝细胞质膜中分离纯化这一共生因子，并寻找其作用部位。1.纯化按Hubbard等人的方法部分纯化大鼠肝细胞质膜胶原酶消化法提取共生活性，但产量太小，不能用于进一步的电泳法和柱层析法纯化。虽然用丙酮、正丁醇或CHAPS萃取法可以使产率提高到原来的50%，但用离子交换树脂进行柱层析后，大部分活性都消失了。用兔抗上述提取物的抗血清可抑制培养的大鼠肝细胞DNA合成，但免疫印迹分析显示抗血清中有大量条带。2.作用部位(S)质膜增加肝细胞增殖所必需的多胺腐胺的浓度，同时完全消除转化生长因子β诱导的肝细胞DNA合成的减少。大鼠肝切除70%后，6小时后，肝脏的共生活性逐渐降低，24小时后基本消失，31小时后恢复到正常水平。伴生因子可能作用于细胞周期的G1期，与腐胺的产生有关，并在作用过程中被灭活或消耗。

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.