肝肾大脏器移植后MMF与Vit D的相互作用及其机制研究

The MMF metabolism has significant individual difference in the patients after liver and kidney transplantation, the reason of which cannot be completely explained only by genetics data according to previous research findings of our research group. Many research indicated that drug-drug interaction is an important reason. Our earlier studies discovered that 1,25-(OH)2D3 up-regulates the expression of UGT1A8/1A10 which were the important MMF metabolic enzymes.Biological information analysis indicated that up-stream of UGT1A8/1A10 include combining site of Vit D receptor- - Vitamin D responsive element(VDRE),and VDRE can set up the expression of down-stream genes.So we aim to explore wether there is the drug-drug intereaction pathway of Vit D→VDR→VDRE→UGT→MMF. Wether Vit D could accellerate metabolism of MMF agent, and relate to individual difference of MMF metabolism. The question is worth of deeply studying. Therefore, we will study the interference of Vit D on MMF metabolism at molecular level;Verify in the animal model and clincal study;and finally discuss the mechanism of drug-drug interference between Vit D and MMF from the molecular level. Importantly, the proposal is going to elucidate the individuality of MMF metabolism from the view of drug-durg interaction, and more comprehensively explain the reason for individual difference of MMF metabolism in the light of the previous genetic research findings, so as to effective guide clinical medication.

肝肾移植抗排斥药MMF代谢存在显著个体差异，本课题组既往研究发现单从遗传学不能完全解释其原因。许多研究表明药物相互作用是代谢个体差异的重要因素。我们前期发现Vit D上调UGT1A8/1A10表达，生物信息学提示UGT1A8/1A10基因上游包含Vit D受体VDR的结合位点VDRE，VDRE可启动下游基因表达。鉴于UGT1A8/1A10是MMF重要的代谢酶，我们分析是否存在Vit D→VDR→VDRE→UGT→MMF药物相互作用途径？Vit D能否通过该途径加快MMF代谢，从而与MMF代谢个体差异相关？值得深入研究。为此，本课题将在细胞水平研究Vit D对MMF代谢的影响；在动物实验和临床研究中加以验证；最后在从VDRE入手在分子水平探讨两者相互作用的分子机制。本课题着重从药物相互作用角度阐述MMF代谢个体差异机制，与既往遗传学研究结果相结合，更全面阐明其代谢个体差异机理并指导临床用药。

背景与目的：器官移植术后MMF代谢个体间差异巨大，除了遗传学因素外，药物间相互作用也是重要因素。本课题在前期基础上，研究Vit D对MMF代谢的影响，并揭示其相互作用的机制，进而指导器官移植术后MMF个体化用药，减少排斥及药物毒副作用。.实验方法：本课题从体内、体外两个方面，分子、细胞、动物及人体四个层面展开研究。通过Real-time PCR在细胞及动物组织中研究Vit D对MMF代谢酶UGT 1A8/1A10表达的影响；通过LC-MS/MS在体外细胞及人体内Vit D对MMF药代动力学的改变；通过CHIP实验、质粒构建、双荧光素酶实验发现UGT1A8/1A10表达调控相关的VDRE区域；通过Crispr实验验证发现的VDRE区域，揭示Vit D影响MMF代谢的分子机制；并通过Vit D刺激及VDR沉默后基因芯片检测探讨VDR调控的下游基因；动物实验探讨鱼肝油、钙剂等Vit D相关药品对MMF代谢的影响；同时开展了肝脏eQTL研究。.实验结果：Real-time PCR发现Vit D可以促进细胞株及动物肠道粘膜中MMF代谢酶UGT 1A8/1A10的表达，人体肠粘膜中VDR与UGT 1A8/1A10表达量正相关；LC-MS/MS检测发现Vit D加快体外细胞及器官移植术后患者体内MMF代谢；分子实验发现多个调控UGT 1A8/1A10表达的VDRE区域；Crispr验证这些VDRE区域可以促进UGT 1A8/1A10表达；钙剂能轻度提高UGT1A10的表达，鱼肝油对UGT1A10表达没明显差异；芯片检测发现许多受VDR调控基因，并发现许多中国人肝脏eQTL位点。.结论和意义：Vit D与MMF之间存在药物相互作用；Vit D激活VDR，与VDRE结合后正相调控促进UGT 1A8/1A10表达，进而加快MMF代谢；许多器官移植术后患者同时服用Vit D与MMF，这是MMF代谢个体差异的重要原因之一。研究结果提示器官移植术后患者在服药Vit D时MMF应该加量，停用Vit D后MMF应该减量。本课题发现了Vit D与MMF之间的相互作用，并揭示了其相互作用的分子机制。本研究有望指导器官移植术后MMF个体化用药，减少排斥并避免药物毒副作用。研究成果发表在Translational Research、Journal of Medical Genetics等杂志。