IL-17基因多态性对肝移植受者环孢素代谢和药效的影响及机制研究

The insufficient or excessive immunosuppression caused by the large individual variability of cyclosporine is the main factor that affects prognosis in transplant patients. Genetic factors are important reasons for individual variability. Recent domestic and international researches have shown that cytokines make the difference of post-transplant immunosuppression, and regulate the expression and activity of cyclosporine metabolic enzymes CYP3A. However, there are few integrated metabolism and efficacy study of twofold effects of cytokine gene polymorphisms on cyclosporine individual variability. Some significant cytokines have been genotyped in transplant patients in our previous studies, and IL-17 gene polymorphisms associated with the individual variability of cyclosporine metabolism and the occurrence of rejection has been found. To our knowledge, IL-17 gene promoter has NFAT-binding domains, and the NFAT is the target gene of cyclosporine. Taken together, we hypothesize the roles of IL-17 gene polymorphisms have two aspects involving that alters drug metabolism by interacting with CYP3A and meanwhile influences efficacy through NFAT, which lead to the individual variability of cyclosporine. In this project, the correlation between IL-17 genotype and individual variability of cyclosporine metabolism and rejection will be studied by first application of sequencing technology, haplotype and NONMEM comprehensive analysis. The effects of IL-17 genotype on expression and activity of CYP3A4 and CYP3A5 metabolism enzymes, and the molecular mechanism related with pregnane X receptor will be explored by methods such as site-directed mutagenesis and gene transfection, etc. Finally, the mechanisms of IL-17 genotype influence cyclosporine efficacy will be elucidated by means of luciferase reporter gene, EMSA, etc. The goal of this project is to search for a new gene marker for individual cyclosporine therapy in liver transplant patients.

环孢素个体差异大导致的免疫抑制不足或过度是影响移植患者预后的主要因素，遗传因素是造成个体差异的重要原因。最近研究表明细胞因子影响移植免疫，调控环孢素代谢酶CYP3A，但从药物代谢及药效两方面综合研究细胞因子基因多态性与环孢素个体差异鲜有报道。我们前期研究提示肝移植受者中IL-17基因多态性与环孢素代谢和药效差异均有相关性。已知IL-17基因启动子存在环孢素靶基因NFAT的结合区域。据此认为IL-17基因多态性在两个层面：即通过CYP3A影响代谢、NFAT影响药效，造成环孢素个体差异。本项目应用测序、单倍型和NONMEM模型综合分析患者IL-17基因型与环孢素代谢个体差异及排斥反应的关联；应用定点突变等技术研究IL-17基因型对CYP3A4/5表达和活性的影响及分子机制；采用荧光素酶报告基因等探讨IL-17不同基因型介导药效差异的作用机理。旨在为肝移植患者环孢素个体化治疗寻找新的基因标志物。

环孢素个体差异大导致的免疫抑制不足或过度是影响移植患者预后的主要因素，遗传因素是造成个体差异的重要原因。课题组前期研究提示肝移植受者中细胞因子基因多态性与环孢素代谢和药效差异均有相关性。本课题主要研究细胞因子IL-17基因多态性对肝移植受者环孢素代谢和药效的影响及调控机制。研究共考察了106例肝移植受者IL-17 G-197A基因型、IL-17血清蛋白水平、有无排斥反应发生等指标，通过萤光素酶测定评估rs2275913 SNP的功能相关性。用IL-17重组蛋白和孕烷X受体（pregnane X receptor, PXR）敲低慢病毒，处理L02细胞，然后分别通过qRT-PCR和蛋白免疫印迹检测PXR、细胞色素P450（cytochrome P450, CYP）3A4、CYP3A5和IL-17R的表达。研究结果表明，在发生排斥反应和无排斥反应的患者中IL-17 G-197A分布有显著差异（P<0.05）。急性排斥反应患者的IL-17水平高于无排斥反应患者。环孢素浓度与不同的IL-17基因型相关（P<0.05）。萤光素酶测定显示IL-17 197G基因型比197A基因型具有更高的萤光素酶活性（P<0.01）。IL-17重组蛋白显著增加了PXR、CYP3A4和CYP3A5的表达（P<0.01），IL-17R的表达无明显变化。当PXR下调时，敲除PXR显著抑制CYP3A4和CYP3A5的mRNA表达，但不抑制IL-17R的表达（P<0.01），IL-17重组蛋白对CYP3A4和CYP3A5的表达无影响。综上所述，本研究揭示了IL-17 G-197A与环孢素代谢和肝移植后移植排斥的关系，这可能与CYP3A4和CYP3A5依赖于PXR的上调有关。