受体介导甲胎蛋白对NIH3T3细胞增殖的促进作用

目的:探讨甲胎蛋白(alpha-fetoprotein,AFP)促新生细胞增殖作用的分子机理。方法:从人脐带血中提取的AFP作用于体外培养的NIH3T3细胞;用3H-TdR参入法观察AFP对细胞DNA合成的影响以及放射受体分析法分析NIH3T3细胞膜表面AFP受体分布;观察在AFP作用下细胞内的第二信使物质环磷酸腺苷(cAMP)浓度及依赖cAMP激活的蛋白激酶A(PKA)活性的改变;Westernblot分析AFP对增殖相关的P21ras表达的影响。结果:10～80mg/L的AFP对NIH3T3细胞的DNA合成有明显的促进作用,与对照组比较最大增幅为121.9%;在NIH3T3细胞膜表面存在2种不同解离平衡常数(KD)的AFP受体,KD1=2.722nmol/L(12810位点/细胞);KD2=89.305nmol/L(119700位点/细胞);AFP能显著提高NIH3T3细胞内的cAMP浓度及PKA活性;对P21ras的表达也有明显的促进作用。结论:AFP促NIH3T3细胞增殖是通过细胞膜表面受体介导,经跨膜cAMP-PKA信号转导途径,影响基因表达来实现的。

ENHANCED THE PROLIFERATION OF ALPHA-FETOPROTEIN ON NIH3T3 CELLS MEDIATED BY RECEPTOR

Objects:In order to investigate the molecular mechanism of alpha-fetoprotein(AFP)on the enhancement of the proliferation of neoplastic cells.Methods :AFP which purified from human cord blood was administrated into the cultured NIH3T3cells in vitro.The effects of AFP(10～80mg /L)on the DNA synthesis was observed by 3 H-TdR incorporation.Using radioactive ligand-receptor binding assay the receptors in the cells;Western blot assay the expression of P21 ras .Results:It was showed that AFP could increase the incorporation of 3 H-TdR into the NIH3T3cells,enhanced ratio was121.9%compared with control.There were two kinds of AFP receptors on the membrane of the cell.The different equilibrium dissociation constanst (KD):KD 1 =2.722nmol/L(12810sites /cell),KD 2 =89.305nmol/L(119700sites /cell)in the membrane of NIH3T3cells.The result also showed that AFP could remarkably improve the activity of protein kinase A (PKA)and increase the level of cyclic adenosine monophosphate(cAMP).Western blot assay demonstrated that AFP could enhance the expression of P21 ras .But the monoclone antibody of AFP can block this function.Conclusions:The growth-regulating effects of AFP on NIH3T3cells are mediated by AFP receptor and which through the transmembrane cAMP-PKA signal transduction pathway to influence the expression of oncogenes.