核受体家族对胎盘ABC膜转运蛋白的转录调控网络研究

With the progress of fetal medicine and perinatology, individualized pharmacotherapy in pregnancy has become a major clinical issue. Individualized pharmacotherapy requires proper transplacental transfer rates for balancing the drug efficacy and toxicity. Our previous studies have demonstrated that the placental ABC membrane transporters play an important role in controlling the transplacental transfer rates. It is of great significance to investigate the transcriptional regulations of placental ABC membrane transporters for identification of novel therapeutic targets, which can be used for individualized pharmacotherapy in pregnancy. Recently, the nuclear receptors have become a focus in drug development. The target genes of nuclear receptors include a variety of drug membrane transporters, such as ABCB1, ABCG2 and ABCC1-5 genes. In our previous work, we performed transcriptional factor binding sites prediction of human ABCB1, ABCG2 and ABCC1-5 genes using bioinformatics analysis. The results implied that there were sets of distinct nuclear receptor binding sites in the promoter regions of ABCB1, ABCG2, and ABCC1-5 genes. In this project, we will study the transcriptional regulations of nuclear receptors on placental ABCB1, ABCG2 and ABCC1-5 genes and elucidate the regulatory network. Moreover, we will identify the novel therapeutic targets for switching the placental barrier. Our study will provide clues and theoretical basis for individualized pharmacotherapy in pregnancy.

随着胎儿医学及围生医学的迅速发展，妊娠期合理安全及个体化用药已成为临床关注的重大课题，合理调控药物胎盘转运率是平衡妊娠期药物疗效与毒副作用的关键点。课题组前期研究结果证实胎盘ABC膜转运蛋白对药物胎盘转运率起着重要调控作用。因此，有必要进一步研究胎盘ABC膜转运蛋白的基因表达调控，找到调控药物胎盘转运率的关键靶点。核受体家族作为近年药物靶点开发研究的热点，其靶基因涉及多种药物膜转运蛋白，其中包括ABCB1、ABCG2及ABCC1-5基因。我们前期使用生物信息学方法对人上述基因启动子区域转录因子结合位点的预测结果亦证实：此类基因启动子区域均存在多个不同核受体结合位点。本项目拟通过研究胎盘ABCB1、ABCG2及ABCC1-5基因的转录调控，阐明核受体与胎盘这些基因的特异转录调控网络，揭示可合理开关血胎屏障的特异性干预靶点，为探讨妊娠期合理安全及个体化用药提供新思路及理论依据。

随着胎儿医学及围生医学的迅速发展，妊娠期合理安全及个体化用药已成为临床关注的重大课题，合理调控药物胎盘转运率是平衡妊娠期药物疗效与毒副作用的关键点。课题组在前期研究的基础上，通过转录因子数据库TRANSFAC8.1，筛选人ABCB1、ABCG2 及ABCC1-5基因启动子上游2000bp至下游500bp区域核受体结合位点，并在Bewo细胞予以验证。进一步经ChIP证实，核受体VDR可特异性地结合ABCB1启动子区域影响基因表达，采用转录因子结合位点点突变技术及双荧光素酶报告系统，基因过表达及RNAi 方法，验证ABCB1基因转录起始位点上游2511至2525bp处（GGGGCGGGGGGGTGC）是VDR在启动子区域特异性结合并产生调控效应的位点，下调VDR表达后，ABCB1基因及蛋白表达均下降，呈正相关；抑制胎盘细胞VDR表达，可以导致P-gp外排功能下降；而过表达VDR对ABCB1基因及蛋白表达以及外排功能均无影响。利用TALEN技术剪切目标基因Vdr的编码区，得到Vdr基因敲除小鼠模型，进一步在体内验证VDR对胎盘ABCB1基因表达及功能的影响，结果表明，VDR基因敲除小鼠在邻苯二甲酸二乙酯（DEHP）暴露下，心脏畸形率明显增加。以上结果阐明了核受体VDR对胎盘ABCB1基因的特异转录调控机制，揭示了可合理开关血胎屏障的潜在干预靶点，为探讨妊娠期合理安全及个体化用药提供新思路及理论依据。