肝刺激因子诱导BEL-7402肝癌细胞促分裂原活化蛋白激酶磷酸化

Phosphorylation of hepatic stimulator substance on mitogen-activated protein kinase in BEL-7402 hepatoma cells

目的 观察肝刺激因子（ｈｅｐａｔｉｃ ｓｔｉｍｕｌａｔｏｒ ｓｕｂｓｔａｎｃｅ， ＨＳＳ）对肝细胞酪氨酸蛋白激酶介导信号传导的调节作用。方法 自鼠肝提取ＨＳＳ，纯化后作用于ＢＥＬ－７４０２肝癌细胞，检测ＨＳＳ对促分裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ， ＭＡＰＫ）活性的影响。结果 生物化学方法提取ＨＳＳ与基因重组ＨＳＳ分子量一致；ＨＳＳ可活化ＭＡＰＫ，其作用强度不及表皮生长因子（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ， ＥＧＦ）；以ＰＤ９８０５９（０～１００ μｍｏｌ／Ｌ）阻断ＭＡＰＫ信号通路后，ＨＳＳ刺激ＭＡＰＫ的作用逐渐减弱，呈剂量－效应关系。结论 ＨＳＳ对酪氨酸激酶信号分子ＭＡＰＫ活性具有调节作用，可能为其促肝细胞增殖的重要机制之一。

Objective To obtain more insight into information of signal transduction of EGF-receptor-mediated pathway response to the stimulation of hepatic stimulator substance (HSS). Methods HSS was extracted from weanling rat liver and partially purified. Bioactivity of HSS was confirmed with its ability to proliferate hepatoma cell in vitro. Meanwhile, a rat recombinant HSS vector was constructed and expressed in BL-21 E. Coli. Mitogen-activated protein kinase (MAPK) activation marked by phosphorylation at Thr202/Tyr204 was determined by Western blot. Results The molecular weight of the biochemically purified HSS was found identically to that of the recombined HSS as expressed in the prokaryotic cells. After the treatment of HSS, cellular MAPK phosphorylation was initiated obviously at 15 min and maintained to 30 min. In comparison with EGF, MAPK phosphorylation as stimulated by HSS appeared less intensive and later time- kinetics as well. The HSS induction on cellular MAPK phosphorylation was gradually inhibited by PD98059, a specific inhibitor of MAPK kinase (MEK). A complete blockade was seen at 100 μ mol/L of PD98059. Conclusions The involvement of HSS on MAPK activation implies that this liver-specific growth factor might take part in, either individually or as combined with other growth factors, the regulation of TPK signaling cascade during hepatocyte proliferation.