肝损伤修复基因IL-6对肝移植术后早期他克莫司代谢的影响及其机制研究

The previous study by our research group has indicated that interleukin 6 (IL-6) gene polymorphisms were associated with the tacrolimus (TAC) concentration/dose ratios in the early period after liver transplantation, which demonstrated that IL-6 may affect TAC metabolism. Drug metabolism enzymes like cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5) and liver function are two main factors affecting TAC metabolism. We have not found IL-6 had obvious regulatory role on CYP3A5 in our preliminary experiments, however, IL-6 polymorphisms were associated with the recovery of liver function. Literatures in recent years have reported that IL-6 played a vital role in liver regeneration by activation of STAT3 pathway which regulated gene transcription related of proliferation and apoptosis. Donor liver regeneration and repair was an important pathological process in the early period after liver transplantation. Therefore, we speculate that there is a progressive mechanism called "IL-6 expression → STAT3 pathway activity → liver regeneration → TAC metabolism" in the early period after liver transplantation. The study intends to build a population pharmacokinetic model to analyze the association between IL-6 polymorphisms and TAC apparent clearance (CL/F). The study also intend to study the influence of IL-6/STAT3 pathway on TAC metabolism using the technology like cell culture, site-directed mutagenesis, gene transfection and rat liver transplantation model. The mechanism will also be verified using SOCS3 protein which negatively regulates IL-6/STAT3 pathway. We aim to find new predictive markers for individual immunosuppressive therapy from the aspect of liver regeneration in the early period after liver transplantation.

课题组前期数据显示供体IL-6基因多态性与肝移植术后早期他克莫司（TAC）浓度/剂量比值关联，提示IL-6影响TAC代谢。药物代谢酶CYP3A5和肝功能是影响TAC代谢的主要因素。我们预实验中IL-6对CYP3A5无明显调控作用；IL-6基因型与肝功能恢复相关。近年文献报道，IL-6通过激活STAT3调控增殖、凋亡相关基因转录在肝再生中起关键作用；供肝损伤后再生修复是肝移植术后早期重要病理过程。因此推测肝移植早期存在"IL-6表达→STAT3通路活性→肝再生→TAC代谢"逐层影响机制。本项目拟构建群体药代动力学模型深入解析IL-6基因型与TAC清除率（CL/F）的相关性；采用细胞培养、定点突变、基因转染、大鼠肝移植模型等技术研究IL-6/STAT3通路对TAC代谢的影响；并应用IL-6/STAT3通路抑制蛋白SOCS3进行机制验证。旨在从肝再生角度为肝移植早期个体化免疫抑制提供新标志物。

背景：他克莫司(TAC)是肝移植患者应用最广泛的免疫抑制剂，其存在治疗窗狭窄、个体间变异性大等特点。实现他克莫司个体化免疫抑制治疗对减少肝移植患者排斥发生、药物不良反应等，提高移植受体长期存活率至关重要。TAC在体内主要通过CYP3A5代谢。IL-6、TLR4等细胞因子调控CYP酶系，还参与肝移植术后排斥反应以及肝脏损伤后肝再生修复，另外，NR1I3等核受体是调控药物代谢酶的重要因素，因此细胞因子和核受体可能通过调控肝功能或CYP酶而影响TAC代谢。.方法：采用Sequenom MassARRAY SNP基因分型技术检测肝移植供、受体CYP3A5、CYP3A4、IL-6、TLR4及NR1I3等基因上的单核苷酸多态性（SNP），收集移植术后他克莫司浓度/剂量(C/D)值、血常规等临床信息；应用免疫组织化学和荧光定量PCR技术检测供肝IL-6表达情况；定点突变技术构建IL-6 rs1800796位点不同基因型LO2肝细胞株，并建立肝细胞缺氧复氧模型，采用ELISA试剂盒、细胞凋亡试剂盒检测细胞活性、增殖，采用western blot、荧光定量PCR方法检测IL-6、CYP3A5、STAT3表达情况情况。分析目的基因的SNP与他克莫司C/D值相关性，以及IL-6基因 rs1800796位点SNP对IL-6/STAT3通路、CYP3A5表达的影响。.结果：供、受体CYP3A5基因多态性与他克莫司代谢的关联得到验证。供体IL6 rs1800796、TLR4 rs1927907、NR1I3 rs11265572及受体CYP3A4 rsl2333983位点SNP与TAC代谢相关。根据IL-6基因rs1800796位点基因型分CC组（快代谢组）和CG+GG组（慢代谢组），快代谢组IL-6在蛋白和mRNA水平都高于慢代谢组；在细胞缺氧复氧模型中发现该位点碱基突变（C→G）后降低肝细胞表达IL-6，降低LO2细胞活性和胞内IL-6、STAT3和CYP3A5的表达。.结论：本项目新发现供体TLR4 rs1927907、NR1I3 rs11265572及受体CYP3A4 rsl2333983位点 SNP与TAC代谢有关；体外实验发现IL-6 rs1800796位点SNP经IL-6\STAT3信号通路实现对肝移植术后早期TAC代谢的影响。将有助于实现肝移植术后他克莫司个体化免疫抑制治疗。