摘要：從雄性初斷乳SD大鼠肝勻漿中提取肝刺激因子(HSS)并加以部分純化, 觀察其促人肝癌細(xì)胞增殖活性及對(duì)p21ras蛋白表達(dá)的影響。結(jié)果表明: (1) HSS具有明顯的促人肝癌細(xì)胞增殖活性, 其分子量為14～20 kD; (2) HSS可提高p21ras蛋白表達(dá), 具有時(shí)間-效應(yīng)關(guān)系, 并與EGF呈協(xié)同作用; (3)HSS調(diào)節(jié)p21ras蛋白表達(dá)具有劑量-效應(yīng)關(guān)系, 且呈現(xiàn)出飽和性。鑒于我們已報(bào)道HSS上調(diào)EGF受體蛋白和基因表達(dá)這一事實(shí), 本實(shí)驗(yàn)結(jié)果進(jìn)一步說明, HSS促人肝癌肝細(xì)胞增殖與其調(diào)節(jié)EGF受體介導(dǎo)的信號(hào)分子傳導(dǎo)過程相關(guān)。

關(guān)鍵詞：肝刺激因子；p21ras；肝臟再生

Abstract:The hepatic stimulator substance (HSS) was partially purified from schedule-lighted weanling rat livers. The effects of HSS on p21ras expression of human hepatic carcinoma cell BEL-7402 were examined. The results showed: (1) HSS was capable of promoting the proliferation of the cells, and its molecular weight was 14～20 kD; (2) the p21ras expression was increased under HSS effect time-dependently, and the effect of HSS appeared synergistic with EGF; (3) the p21ras expression induced by HSS manifested a dose-dependent manner, and the saturation effect of HSS occurred at the concentration of 100 μg/ml. Considering together with our previous report about HSS regulation on EGF receptor, these results strongly suggest that the proliferatory effect of HSS on hepatic carcinoma cells is presumedly related with EGF receptor-mediated signal transduction.

Key words:hepatic stimulator substance; p21ras; liver regeneration

肝臟具有強(qiáng)大的再生能力。肝再生是在多因素參與下有序的調(diào)節(jié)過程［1～3］。表皮生長因子(EGF)、轉(zhuǎn)化生長因子α(TGF-α)、肝細(xì)胞生長因子(HGF)、胰島素等均具有顯著的促進(jìn)肝細(xì)胞增殖的作用。然而, 這些生長因子不具有組織特異性, 因此僅從這些因子出發(fā)難以闡明肝再生調(diào)節(jié)的分子機(jī)制。LaBrecque于1975年首先從初斷乳大鼠肝中提取肝再生刺激因子(hepatic regenerative stimulatory substance, HRSS［4］, 后稱HSS), 繼而研究了HSS的理化特征［5～8］。 HSS可刺激肝細(xì)胞DNA合成, 促進(jìn)肝細(xì)胞由G1期進(jìn)入S期［9］; 穩(wěn)定細(xì)胞膜, 減輕CCl4和半乳糖胺對(duì)肝細(xì)胞的毒性作用［10,11］。體外研究證明, HSS確為人胎肝細(xì)胞基因表達(dá)產(chǎn)物［12］。盡管人們一直努力將HSS制成純品［7, 13］,但未見成功報(bào)道。鑒于HSS能增強(qiáng)EGF促肝細(xì)胞增殖活性, 近年來也有人將HSS稱之為肝再生增強(qiáng)因子(augmentor of liver regeneration, ALR)［14］。HSS是否與已克隆出的ALR同為一體, 尚不能zui終定論。 我們曾報(bào)道, HSS促肝細(xì)胞增殖活性與EGF受體表達(dá)相關(guān)［15,16］。為研究HSS對(duì)EGF受體介導(dǎo)下游信號(hào)傳導(dǎo)的調(diào)節(jié)作用, 本實(shí)驗(yàn)觀察了HSS對(duì)人肝癌細(xì)胞系BEL-7402 p21ras表達(dá)的影響, 以進(jìn)一步探討HSS作用的可能機(jī)制。

1.材料和方法

1.1 實(shí)驗(yàn)動(dòng)物[17] 雄性初斷乳SD大鼠(60～90 g), 節(jié)律光照飼養(yǎng)1周(7∶00～19∶00光照, 19∶00～次日7∶00避光), 自由飲食。

1.2 HSS的提取及部分純化［18］將上述SD大鼠于第8日7∶00～9∶00之間斷頸處死, 剖腹摘取肝臟, 制備HSS。將HSS濃縮(CT60, 丹麥Heto生產(chǎn))至2 ml, 經(jīng)Sephadex-G75(Superfine, 瑞典Pharmacia公司)層析, 流動(dòng)相為DPBS(pH7.4), 洗脫速度為35 ml/h。以分光光度計(jì)(Lamda-Bio, 美國PE公司)測定流分體積的吸光值(OD280), 并以此為橫坐標(biāo), OD280為縱坐標(biāo)繪制蛋白的分離色譜圖。