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为探讨甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞增殖的促进作用。用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,通过MTT计数、[3 H] TdR参入法、流式细胞仪等研究细胞增殖 ,并用放射免疫法测定细胞内cAMP、共聚焦显微镜扫描细胞内Ca2 + 浓度等观察细胞内第二信使物质的改变及测定3 2 P转移反映细胞内蛋白激酶A(PKA)活性 ;用Western印迹分析法分析突变型 p5 3、p2 1ras蛋白表达。结果显示AFP(浓度大于 10mg/L)对HeLa细胞增殖有明显的促进作用。在AFP(2 0mg/L)作用下 ,HeLa细胞cAMP浓度升高 30 0 % ;Ca2 + 浓度最大升高 15 4 .9%、PKA活性最大升高 10 0 %。突变型 p5 3和 p2 1ras分别增高 81.1% (2 4h)和 96 .2 % (2 4h)。抗甲胎蛋白单克隆抗体能有效地阻断AFP促HeLa细胞增殖作用。以上表明 ,(1)AFP具促HeLa细胞增殖的生理功能 ;(2 )AFP促进HeLa细胞增殖的作用是通过影响细胞内cAMP和Ca2 + 浓度以及PKA活性进而促进一些原癌基因的表达来实现的。
To investigate the role of alpha fetoprotein (AFP) on tumor cell proliferation. HeLa cells cultured in vitro were treated with AFP extracted from human umbilical cord blood and cell proliferation was examined by MTT counting, [3 H] TdR incorporation, flow cytometry and the like, and intracellular cAMP was determined by radioimmunoassay. Confocal microscopy The intracellular Ca2 + concentration was measured by scanning the intracellular Ca2 + concentration. The changes of intracellular second messenger were observed. The 3 2 P transference reflected the activity of protein kinase A (PKA) in cells. The expression of mutant p5 3 and p2 1ras proteins was analyzed by Western blot analysis. The results showed that AFP (concentration> 10mg / L) significantly promoted the proliferation of HeLa cells. Under the action of AFP (20 mg / L), the concentration of cAMP in HeLa cells increased by 30%, the maximum Ca2 + concentration increased by 15.49%, and the maximum PKA activity increased by 10%. Mutant p5 3 and p2 1ras increased by 81.1% (24 h) and 96.2% (24 h), respectively. Anti-AFP monoclonal antibody can effectively block the proliferation of HeLa cells stimulated by AFP. The above shows that: (1) AFP has the physiological function of promoting proliferation of HeLa cells; (2) AFP promotes the proliferation of HeLa cells through promoting the expression of some proto-oncogenes by affecting the intracellular cAMP and Ca2 + concentrations and PKA activity .