磷酸化流式技术评估肝移植后P70S6激酶活性的意义

Assessment of P70S6 kinase phosphorylation after liver transplantation by phospho-flow cytometry

目的 探讨应用流式磷酸化技术检测肝移植受体外周血CD3＋细胞的mTOR通路下游P70S6激酶的磷酸化水平及价值.方法 选取自2008年10月至2013年10月首都医科大学附属北京朝阳医院肝移植患者84例,其中以西罗莫司为主要免疫抑制剂方案的26例（西罗莫司组）,以他克莫司为主要免疫抑制剂方案的35例（他克莫司组）,以环孢素A为主要免疫抑制剂方案的23例（环孢素A组）.检测各组患者CD3＋细胞P70S6激酶磷酸化水平,分析西罗莫司组P70S6激酶磷酸化水平与血药浓度的相关性.同时检测3例健康人P70S6激酸磷酸化程度的个体内差异和个体间差异.结果 健康人P70S6激酶磷酸化个体内差异的变异度范围为3.5％ ～ 5.6％,个体间差异变异度为18.9％ ～22.5％.西罗莫司组CD3＋细胞P70S6激酶磷酸化（28.9±10.5）显著性低于健康对照组（57.2±8.4,P＜0.001）,他克莫司组（42.5±14.1,P＜0.001）和环孢素A组（51.4±10.9,P＜0.00l）.健康对照组的P70S6激酶活性（57.2±8.4）显著性高于他克莫司组（42.5±14.1,P＜0.01）和环孢素A组（51.4±10.9,P＜0.05）.在西罗莫司组,P70S6激酶磷酸化与血药浓度无关（r=-0.18,P=0.39）.结论 磷酸流式技术可以通过检测P70S6激酶磷酸化评估mTOR的抑制程度,检测P70S6激酶磷酸化水平可以为个性化免疫治疗提供更好的依据.

Objective To explore the role of P70S6 kinase phosphorylation as a downstream of mammalian target of rapamycin （mTOR） pathway in CD3 positive cells of liver transplant patients.Methods A total of 84 liver transplant recipients were recruited from our hospital and divided into 3 treatment groups of sirolimus （n =26）,tacrolimus （n =35） and cyclosporine （n =23）.The P70S6 kinase phosphorylation of CD3 positive cell of patients and healthy control （HC） were analyzed by phospho-flow cytometry.A correlation analysis between P70S6 kinase phosphorylation and sirolimus trough level was performed.Intra-individual variability and inter-individual variability of P70S6 kinase phosphorylation were measured.Results The P70S6 kinase phosphorylation in HC showed a low degree of intra-individual variability （3.5％ to 5.6％） while the inter-individual variability between different healthy volunteers was higher （18.9％ to 22.5％）.The P70S6 kinase phosphorylation in patients treated with sirolimus （28.9 ± 10.5） was significantly lower than in HC （57.2 ± 8.4,P ＜ 0.001）,tacrolimus （42.5 ± 14.1,P ＜ 0.001） or cyclosporine treated ones （51.4 ± 10.9,P ＜ 0.001）.The P70S6 kinase phosphorylation in HC （57.2 ± 8.4）was significantly higher than in tacrolimus （42.5 ± 14.1,P ＜ 0.01） or cyclosporine treated patients （51.4 ± 10.9,P ＜ 0.05）.No correlation existed between P70S6 kinase phosphorylation and trough level of sirolimus （r =-0.18,P =0.39）.Conclusion Phospho-flow cytometry assay has been established for determining the degree of mTOR inhibition by assessing P70S6 kinase phosphorylation.Quantification of P70S6 kinase phosphorylation may play an adjunct role in pharmacodynamically guiding an individualized mTOR inhibitor based immunosuppression.