基于纳米技术的超微量蛋白免疫分析ERK的磷酸化

Nanofluidic Proteomic Immunoassay of Phosphorylation of ERK

目的运用基于纳米技术的超微量蛋白免疫分析(Nanofluidic proteomic immunoassay,NIA)方法检测ERK的磷酸化。方法构建尾加压素Ⅱ(UrotensinⅡ)受体GPR14细胞系,UⅡ(10-7M)刺激该细胞系10分钟后,提取细胞蛋白采用传统Western方法和NIA技术分别检测同种样品ERK的磷酸化,并用两种方法观察UⅡ受体拮抗剂Urantide对ERK磷酸化的抑制作用。通过结扎C57BL/6小鼠冠状动脉左前降支,建立心梗模型,假手术组开胸不结扎,术后1周通过流式细胞术分离心脏侧群干细胞(Cardiac side population cells,CSPs),用NIA技术检测ERK磷酸化。结果 Western分析可观察到UⅡ介导ERK1/2的磷酸化,Urantide抑制该效应。NIA分析可观察到UⅡ介导ERK1/2磷酸化的多种异构体包括p-ERK1a,P-ERK1b,PP-ERK1,PP-ERK2水平升高,Urantide同样抑制该效应。心梗后CSPs数目明显增加。NIA分析显示,心梗促进CSPs上的p-ERK1b,PP-ERK1的水平升高,但对PP-ERK2影响不大。结论通过GPR14细胞系的分析,NIA技术灵敏度高,可检测ERK磷酸化的多种形式,运用该技术可检测数万个CSPs的ERK磷酸化,可观察心梗促进CSPs的某些ERK磷酸化异构体的水平升高,提示可能与心梗后CSPs的增殖有关。

Objective To measure phosphorylation of ERK by Nanofluidic proteomic immunoassay （NIA）. Methods Urotensin II （UII） receptor GPRI4 cell line was constructed and stimulated by UII（10-7M） with or without Urantide for 10 min, cells were lysed and ERK activation was determined by western blot or NIA respectively. Mice myocardial infarction （MI） model was produced by ligating the left anterior descending coronary artery. Sham operation was used as control. Cardiac side population cells （CSPs） were isolated by fluorescence-activated cell sorting （FACS） at 1 week after MI or Sham to analyze the phosphorylation of ERK1/2 by NIA. Results By western blot and NIA analysis, UII increased the phosphorylation of ERK1/2 and urantide inhibited these effect in GPR14 cell line. NIA analysis could determine more phosphorylated isoforms of ERK such as p-ERKla, p-ERKlb, pp-ERKI, pp-ERK2 than western blot. MI induced the proliferation of CSPs. NIA analysis of a few ten thousands CSPs indicated that MI promoted the increase in p-ERKIb and pp-ERK1 level but had little effect on pp-ERK2. Conclusions We described a new and highly sensitive method -NIA for determining multiple phosphoisomers of ERK in GPR14 cell line. NIA analysis of several ten thousands CSPs revealed that MI increased the level of phosphorylated isoforms of ERK1/2 which may be involved in the proliferation of CSPs after MI.