甲胎蛋白促人肝癌Bel7402细胞增殖的机制研究

SIGNAL TRANSDUCTION MECHANISM OF ALPHA-FETOPROTEIN PROMOTING PROLIFERATION OF HUMAN HEPATOMA Bel7402 CELLS

目的:观察甲胎蛋白促肿瘤细胞增殖的信号转导方式。方法:用从人脐带血清中提取的甲胎蛋白,作用于体外培养的人肝癌Bel7402细胞,运用MTT染色计数法,流式细胞仪测定细胞周期和3H-脱氧胸腺嘧啶核苷(3H-TdR)参入法等指标检测细胞增殖状况;放射受体分析细胞膜上甲胎蛋白受体存在和数目;免疫结合法检测细胞内cAMP浓度;Westernblot分析P21ras蛋白的表达。结果:甲胎蛋白(10～80mg·L-1)作用24h后,人肝癌Bel7402细胞有不同程度的促增殖作用;在人肝癌Bel7402细胞膜上存在2种不同的解离平衡常数(Kd)的甲胎蛋白受体,Kd分别为Kd1=1.3×10-9mol·L-1(89400位点/细胞)和Kd2=9.9×10-8mol·L-1(582000位点/细胞)。AFP能显著提高Bel7402细胞内的cAMP浓度,对P21ras的表达也有明显的促进作用。甲胎蛋白的单克隆抗体能阻断这种作用。结论:甲胎蛋白具有促人肝癌Bel7402细胞增殖的作用,这种作用是由受体介导的cAMP信号转导途径实现的。

Objective:To observe the signal transduction mechanism of alpha-fetoprotein(AFP)promoting proliferation of tumor cells.Method:Used AFP which purified from human cord serum,act on human hepatoma Bel7402cell which cultured in vitro.Utilized the MTT counts,cell cycle phase analysis with flow cytomtry and 3 H-thymidine deoxyribonucleotide( 3 H-TdR)incorporation.With radioactive labeled 125 I-AFP for receptor binding assay,Utilized radioactive immunosorbent assay to detect cAMP accumulation,and the expression of P21 ras protein were analyzed by Western blot.Results:AFP (10-80mg ·L -1 )can enhance the proliferation of Bel7402cells after incubating them with AFP for24hours;There two subtypes of AFP receptors were identified in cells,with the Kds of Kd 1 =1.3×10 -9 mol·L -1 ,Kd 2 =9.9×10 -8 mol·L -1 in Bel7402cells.Pretreatment of cells with AFP resulted in significant increase in cAMP accumulation;Western blot assay demonstrated that AFP could enhance the expression of P21 ras of the cells;but the monoclone antibody of AFP can block these function.Conclusion:AFP can enhance the proliferation of some neoplastic or tumor cells and this effect was mediated by AFP receptor,the receptor mediated was by intracellular cAMP -signal transduction pathway.