维甲酸类似物抑制PI3K/Akt信号通路逆转早幼粒白血病细胞维甲酸耐药的分子机制

The main reason of relapse and refractory patients in acute promyelocytic leukemia (APL) are recognized as resistance to all-trans retinoic acid (ATRA). According to resent study, ATRA-resistance of APL cells show aberrant up-regulation PI3K/AKT signaling activity,A new ATRA-like analogue which modified structure of tretinoin and Added to benzene ring can reverse ATRA resistance, The detailed mechanisms are not fully understood.In previous study, I found that this ATRA-like analogue could induce NB4-resistance-ATRA-cell (NB4RA) early apoptosis, decreased the level of PI3K and Akt protein, and those were linear correlation between the concentration of ATRA-like analogue and PI3K and Akt protein level, The molecule mechanisms is unclear.It will indicate direction and evidence of modification tretinoins's structure to reverse ATRA resistance if we can understand the effection site and regulation gene of ATRA-like analogue to ATRA resistance cell.We are ready to use mass spectrography to validate bind relationship between ATRA-like analogue and PI3K, Nuclear Magnetic Resonance and molecular mimicry determine the bind subuniet and site of interspace conformation.Then we use the specific PI3K bind site inhibitors to inhibite PI3K/Akt signaling pathway to study how AM80 effect cell growth, differentiation and apoptosis of NB4RA, gene chips to screening downstream gens, PCR and Western blotting to validate gene and protein respectively. we repeat experiment about those positive conclusions with ATRA resistance APL patient of leukemia cells.This research conclution will not only deeply understand the molecular mechanisms of APL resistant mechanism, but also provide theory foundation how to modify tretinoins's structure to reverse ATRA resistance, It will be a new idea and methods for solving APL resistant treatment.

维甲酸耐药是急性早幼粒细胞白血病（APL）治疗失败或复发的主要原因，近期研究表明耐药后PI3K/Akt通路活性升高，改良维甲酸结构添加苯环的类似物能逆转耐药，机制未明。前期研究观察到此维甲酸类似物干预耐药NB4细胞后凋亡提早，PI3K、Akt蛋白量下降，下降水平与维甲酸类似物浓度呈线性关系，调控机理未明。明确其作用位点和调控基因，可为改良维甲酸结构克服耐药提供方向和依据。本课题组拟以质谱法确定维甲酸类似物与PI3K的结合关系，核磁共振与分子模拟确定空间构象结合位点；使用特异性阻滞剂在此位点上阻断PI3K/Akt通路,观察维甲酸类似物如何影响耐药NB4细胞的生长、分化和凋亡，基因芯片筛选下游受调控基因，PCR和蛋白质印迹法确认阳性表达基因和蛋白；以维甲酸耐药患者APL细胞验证上述阳性结果。研究结果将加深认识APL耐药机制，为如何改良维甲酸结构克服耐药提供理论支持，发现解决APL耐药的新思路。