Wang Jun-Yu, Fan Hua
Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.
Chin Med J (Engl). 2015 Mar 5;128(5):664-9. doi: 10.4103/0366-6999.151670.
The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.
Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.
Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).
The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.
p70S6激酶（p70S6K）的磷酸化是西罗莫司药效学效应灵敏检测的一个重要靶点，但评估p70S6K磷酸化的方法仍不明确。本研究旨在通过不同方法研究肝移植患者外周血单个核细胞（PBMCs）中雷帕霉素哺乳动物靶蛋白（mTOR）通路下游的p70S6K磷酸化情况。
本研究分析了首都医科大学附属北京朝阳医院的75例肝移植受者。患者分为三组，接受西罗莫司治疗的患者（n = 22）、接受他克莫司治疗的患者（n = 30）、接受环孢素治疗的患者（n = 23）。采用磷酸化流式细胞术和蛋白质免疫印迹法分析患者及健康对照者（HC，n = 12）PBMCs中p70S6K的磷酸化情况。对磷酸化流式细胞术和蛋白质免疫印迹法的数据进行相关性分析。测定HC及不同患者中p70S6K磷酸化的批内变异。
磷酸化流式细胞术中p70S6K磷酸化的批内变异为4.1%至8.4%，蛋白质免疫印迹法中为8.2%至18%。接受西罗莫司治疗的患者（19.5±7.7）的p70S6K磷酸化显著低于HC（50.1±11.3，P<0.001）、接受他克莫司治疗的患者（37.7±15.7，P<0.001）或接受环孢素治疗的患者（41.7±11.7，P<0.001）。HC（50.1±11.3）的p70S6K磷酸化显著高于接受他克莫司治疗的患者（37.7±15.7，P<0.01）或接受环孢素治疗的患者（41.7±11.7，P<0.01）。磷酸化流式细胞术数据与蛋白质免疫印迹法数据之间存在相关性（r = 0.88，P<0.001）。
通过磷酸化流式细胞术和蛋白质免疫印迹法确定了通过评估p70S6K磷酸化来衡量的mTOR抑制程度。评估p70S6K磷酸化可能在基于免疫抑制的西罗莫司药效学指导和个体化方面发挥辅助作用。
