转运体ABCC4/ABCC5在叶酸拮抗剂耐药现象中的机制研究

叶酸拮抗剂广泛用于临床治疗，耐药和毒副作用是导致疗效不佳的主要原因，但迄今叶酸拮抗剂耐药机制仍不明确。ABC转运体是叶酸拮抗剂排泄中主要转运蛋白，但其在叶酸拮抗剂耐药中的作用缺乏系统研究。我们前期结果显示甲氨蝶呤耐药细胞株中ABCC4、ABCC5表达增加，且对其他叶酸拮抗剂也产生了交叉耐药，但具体机制还需进一步探讨。为此，我们提出假说：ABCC4、ABCC5参与叶酸拮抗剂耐药，尤其是新型叶酸拮抗剂、大剂量叶酸拮抗剂中毒时外排的重要因素。为了验证这一假说，本研究将从HEK293细胞模型、gene-delivery老鼠模型、临床试验等多个层次，采用real time PCR、Western blot、AVV9腺病毒载体感染动物、质粒转染细胞等手段，探讨ABCC4、ABCC5在叶酸拮抗剂耐药中的具体作用机制。本项目将分析ABCC4/5在叶酸拮抗剂PK/PD发挥的作用，为个体化用药提供理论基础。

Antifolates are commonly used in clinical,drug-resistance and toxicity are the main reason of treatment failure. But molecular mechanism of antifolates drug-resistance is still confuse. .ABC transporters play key role in antifolate efflux, but it is short of systematic research there are few study about their role in antifolates drug-resistance..Our pre-experiments showed that expression of ABCC4,ABCC5 were significant increased in the methotrexate-resistant cells, and the IC50 of other antifolates were also increased significantly，but the machanism still unknown. So our hypothesis are:ABCC4/5 can efflux the antifolates, especially high-dose antifolates and new members of antifolates..The goal of this proposal is to test the hypothesis that these two pumps affect antifolates efflux by using HEK293 cells model,gene delivery mouse model, clinical trial.In this experiments, we will using those technical, such as real time PCR, Western blot, Adeno-associated virus (AAV)-mediated gene delivery, plasmid transfection and so on, to explore the molecular mechanism of ABCC4/5 in antifolates drug-resistance.. We will analyze the molecular mechanism of ABCC4,ABCC5 in the pharmacodymacis and phacokenetics of antifolates. We hope will this research could provide a way to increase the effectiveness of antifolates, and thus increase the cure rate in disease.