IL-10、IL-18基因多态性对肺移植患者他克莫司代谢个体差异的影响及其分子机制研究

Rejection, infection and toxicity of immunosuppressive underdosage or overdosage are important factors that effect prognosis in lung transplant patients, and individual therapy is critical strategy. Significant cytokines were genotyped by Sequenom MassArraay SNP Genotyping System in liver transplant patients in our previous studies. Recent research has shown that cytokines regulate expression and activity of drug metabolism enzyme. Expression of cytokines was obviously different in vivo because of variations in interethnic and different organ transplants. A number of previous studies evaluated the interindividual and intraindividual variability of tacrolimus pharmacokinetic in interethnic populations and different organ transplants , similar information is not all available for chinese lung transplant patients because of variations in ethnic and organ transplants. For this reason, influences of IL-10 and IL-18 gene polymorphism on tacrolimus metabolism need to be explored in chinese lung transplant patients. First application of sequencing technology, haplotype analysis and population pharmacokinetics analyzes association between IL-10/ IL-18 genotype and individual difference of tacrolimus metabolism. Second the project studies effect of IL-10 and IL-18 on expression and activity of CYP3A4 and CYP3A5 metabolism enzymes by cell culture and Site-directed Mutagenesis,etc. Finally the project elucidates pregnane X receptor mediating influence of IL-10 and IL-18 on CYP3A4 and CYP3A5 by RNA interference and gene transfection,etc. The goal of this project provides new genetic marker for individual immunosuppressive therapy in lung transplant patients.

肺移植术后免疫抑制不足或过量导致的排斥反应、感染和药物毒副作用是影响患者预后的重要因素，个体化用药是关键解决措施。课题组前期研究应用时间飞行质谱生物芯片系统对肝移植患者重要细胞因子进行基因分型，显示IL-10、IL-18基因多态性与他克莫司代谢个体差异相关。近年研究表明细胞因子调控药物代谢酶表达和活性。由于种族和不同器官移植差异，内环境中细胞因子表达情况明显不同，其基因型对中国肺移植患者他克莫司代谢个体差异影响需深入探讨。本项目应用测序、单倍型和群体药代动力学分析患者IL-10和IL-18基因型与他克莫司代谢个体差异的关联；采用细胞培养、定点突变等技术研究IL-10和IL-18基因型对CYP3A4、CYP3A5 表达和活性的影响；应用RNA干扰、基因转染等阐明孕烷X受体介导IL-10和IL-18对CYP3A4、CYP3A5影响的分子机制。旨在为肺移植患者个体化免疫抑制治疗提供新基因标志物。

研究背景：肺移植是治疗终末期肺病的唯一有效手段。他克莫司（Tacrolimus, FK506）作为一线免疫抑制剂被广泛用于肺移植术后的抗排斥反应。FK506具有治疗窗窄、个体差异大的特点。免疫抑制不足及免疫抑制均导致肺移植患者不良预后，个体化免疫抑制治疗是关键解决措施。FK506主要由CYP3A5代谢。细胞因子调控细胞色素酶表达和酶活性。本项目研究细胞因子白细胞介素-10（IL-10）和白细胞介素-18（IL-18）对肺移植患者FK506药物清除的影响作用。方法：上海市肺科医院住院接受肺移植手术的患者51例为研究对象。MassARRAY平台进行基因分型，包括IL-18 (rs5744247，rs1946518), IL-10 (rs1800896, rs1800872，rs3021097) 和 CYP3A5 rs776746。同时收集肺移植术后1个月C/D（校正后浓度/剂量比值μg/mL/mg/kg）数值。结果：单基因型分析显示IL-18 rs5744247 C和 rs1946518 A 为FK506 快代谢等位基因。快代谢等位基因数目≥3肺移植患者FK506 C/D值明显低于快代谢等位基因数目≤2肺移植患者。在CYP3A5表达的肺移植患者中，IL-18快代谢等位基因数目与FK506 C/D值相关联（P=0.016, 0.043, 0.0069, 0.0059）；CYP3A5不表达的肺移植患者中，IL-18快代谢等位基因数目与FK506 C/D值无关联。IL-10 rs1800896在术后第1、2周可影响FK506代谢（P=0.027和P=0.034）。本组资料中未发现IL-10 rs1800872 和 rs3021097基因型对肺移植患者FK506代谢的影响。肝细胞中IL-18 rs5744247 和rs1946518 影响其mRNA 表达水平 (GG > CG > CC, P=0.0175；CC > CA >AA, P=0.0403)。免疫组织化学检测显示IL-18 在肝细胞表达。IL-18处理肝细胞导致CYP3A5 mRNA表达的不同（P=0.017）。结论：IL-18 rs5744247和 rs1946518基因型影响肺移植患者FK506代谢，与FK506经典标志物CYP3A5 rs776746联合检测，在肺移植精准免疫抑制治疗中具有潜在的应用前景。