中文摘要：

目的 研究甲胎蛋白（AFP）对肝癌细胞内PI3K/AKT信号传递的影响,以及其在癌细胞耐受全反式维甲酸（ATRA）中的作用. 方法 四甲基偶氮唑盐（MTT）法检测ATRA对人肝癌Bel 7402细胞增殖的影响;显微照相观察细胞形态学的改变;流式细胞分析细胞凋亡;激光共聚焦显微镜观察AFP与PTEN的共定位;免疫共沉淀（Co-IP）技术研究AFP与PTEN相互作用;Westemblot法分析磷酸化的蛋白激酶B（pAKT）和Src表达,构建干扰AFP表达的载体（AFP-siRNA）并转染Bel 7402细胞;用PI3K特异性阻断剂Ly294002处理细胞,分析Ly294002的作用效果.通过t检验分析组间的统计学差异. 结果 MTT分析显示,人肝癌Bel 7402耐受ATRA的细胞毒性;共聚焦显微镜观察显示AFP与PTEN共定位于细胞质;Co-IP技术研究发现AFP能与PTEN结合;MTT分析发现,Bel 7402细胞转染AFP-siRNA载体后24 h后,细胞生长显著受抑制,与对照组比较,差异有统计学意义（P＜0.05）,而且AFP-siRNA载体和ATRA共同处理组,细胞生长受到抑制更为显著,与对照组、单独处理组比较,差异均有统计学意义（P＜ 0.01）;干扰AFP表达能显著增加Bel 7402细胞对ATRA敏感性,并能抑制pAKT和Src的表达;Ly294002能抑制AFP促进Bel 7402细胞表达pAKT和Src的作用.结论 肝癌细胞内表达的AFP能与PTEN结合并抑制PTEN对AKT的去磷酸化作用,肝癌细胞内高表达的AFP能激活PI3K/AKT信息通路对抗ATRA诱导的凋亡.

英文摘要：

Objective To explore the effect ofalpha-fetoprotein （AFP） on transduction of the PI3K/ AKT signal in hepatocellular carcinoma cells and the role played by AFP in resistance to cytotoxicity of all-trans retinoic acid （ATRA）.Methods The effects of ATRA of human liver cancer cells was assessed using the BEL-7402 cell line with the MTT assay （to evaluate proliferation）,microscopy （to evaluate morphology）,flow cytometry （to evaluate apoptosis）,laser confocal microscopy and coimmunoprecipitation （co-IP; to evaluate co-localization and interaction of AFP with PTEN）,Western blotting （to evaluate expression of phosphorylated-protein kinase B （pAKT） and Src,and RNA interference （RNAi）-mediated knockdown of AFP.Finally,applicationof the PI3K-specific inhibitor Ly294002 was used to monitor the influence of AFP in transduction of the PI3K signal pathway.Results The human hepatoma cell line BEL-7402 were resistant to ATRA cytotoxicity.PTEN and AFP co-localized in the cytoplasm,and co-IP indicated that AFP interacts with PTEN in BEL-7402 cells.RNAi knockdown of AFP expression led to reduced growth of BEL-7402 cells.BEL-7402 cells transfected with AFP-short interfering （si）RNA vectors showed enhanced sensitivity to ATRA and reduced expression of pAKT（Ser473） and Src; Ly294002 reduced the role of AFP in stimulating expression ofpAKT（Ser473） and Src.Conclusion AFP can activate transduction of the PI3K/AKT signal,and expression of AFP in hepatoma cells is a pivotal event for resisting ATRA-induced apoptosis.