P-糖蛋白转运体对外周血单个核细胞中他克莫司浓度的影响及与肺移植术后慢性排斥反应的量效关系研究

Chronic rejection is one of the important reasons for the low survival rate after lung transplantation. Tacrolimus has the highest use rate in postoperative maintenance therapy. However, the blood components after lung transplantation are complex. Clinical monitoring of tacrolimus whole blood trough concentration may not accurately reflect its pharmacodynamic concentration. It is necessary to monitor tacrolimus concentration in lymphocytes. Previous studies had also found that tacrolimus concentration in whole blood, plasma and blood cells of patients after lung transplantation has a low correlation. Tacrolimus efflux of lymphocyte will be mediated by P-gp transporter encoded by ABCB1. The association between ABCB1 polymorphism and tacrolimus concentration in PBMC of lung transplantation patients is less studied. ABCB1 polymorphism has obvious ethnic differences, so it is necessary to study its characteristics in Chinese population. The correlation between P-gp expression and tacrolimus concentration in PBMC remains to be studied. Therefore, this project aims to analyze the correlation between the expression level of P-gp transporter on PBMC cell membrane and tacrolimus concentration in PBMC. The correlation between ABCB1 polymorphism and tacrolimus concentration in PBMC, the dose-response relationship between PBMC tacrolimus concentration and chronic rejection after lung transplantation will also be studied. Cox regression model was established to analyze the influencing factors of the incidence of chronic rejection in lung transplantation and to improve the survival rate of patients after lung transplantation.

肺移植术后慢性排斥反应是导致肺移植患者生存率不高的重要原因，他克莫司在术后维持治疗方案中使用率最高。然而，肺移植术后血液成分较复杂，临床监测他克莫司全血谷浓度可能无法准确反映其药效浓度，需监测淋巴细胞中他克莫司浓度。本课题前期研究也发现，肺移植术后患者全血、血浆、血细胞中他克莫司浓度相关性较低。淋巴细胞膜上P-gp转运体外排他克莫司，其编码基因ABCB1多态性与肺移植患者PBMC中他克莫司浓度相关性研究较少。ABCB1多态性具有较明显的人种差异，有必要研究中国人群特点。P-gp表达量与PBMC中他克莫司浓度的相关性也有待研究。因此，本项目拟分析肺移植术后患者PBMC细胞膜表面P-gp转运体表达水平、ABCB1多态性与PBMC中他克莫司浓度的相关性，以及PBMC他克莫司浓度与肺移植术后慢性排斥反应的量效关系。建立Cox回归模型，分析肺移植慢性排斥反应发生率的影响因素，提高患者生存率。

肺移植术后血液成分较复杂，临床监测他克莫司全血谷浓度可能无法准确反映其药效浓度，需监测淋巴细胞中他克莫司浓度。PBMC细胞膜中P-gp转运体外排他克莫司，其编码基因ABCB1多态性与肺移植患者PBMC中他克莫司浓度相关性研究较少。本研究利用纳升液相串联高分辨质谱（nanoLC-HRAM）建立新的PBMC中他克莫司浓度及细胞膜中P-gp表达量的高灵敏度检测方法。收集216例肺移植受者，采集患者信息、用药信息，检测PBMC中他克莫司浓度、细胞膜中P-gp表达量、ABCB1基因多态性及血浆IL-2水平。利用Lasso回归筛选变量，通过Log-rank检验、KM曲线、ROC曲线分析PBMC中他克莫司浓度的量效关系、PBMC中他克莫司浓度及肺移植受者排斥反应发生率的影响因素。结果显示，新建的PBMC中他克莫司浓度检测方法全血使用量仅1mL，减轻了患者损伤，细胞计数校正和总蛋白量校正两种校正方法校正结果一致性较好，可通过公式互换。新建的P-gp表达量检测通量较高，细胞膜中P-gp表达量随肺移植受者生存时间延长显著上调，提示肺移植受者可能出现耐药。肺移植受者CPBMC/Dose的影响因素包括年龄、HGB、ALB、移植后时间、3435 G基因型、2677 T基因型、联合用药信息，其多元线性回归模型可以预测CPBMC/Dose变异的63.8%。本研究还发现CPBMC/Dose浓度至少达到0.261 pg/(μg protein•d)，可以将血浆IL-2的浓度抑制在55.87fg/mL以下，并且降低肺移植排斥反应发生率，为PBMC中他克莫司浓度的有效范围提供参考。此外，本研究筛选出肺移植受者排斥反应发生率的影响因素包括年龄、性别、肌酐、PBMC他克莫司浓度（CPBMC）、联合用药信息等，Cox回归模型的C-index值为0.788，预测能力较好。本研究将为临床降低肺移植排斥反应发生率提供线索。