利用荧光生物感受器研究p38MAPK的生物活性

发布时间：2017-12-28 09:35

本文关键词：利用荧光生物感受器研究p38MAPK的生物活性　出处：《生物技术》2014年06期 　论文类型：期刊论文

【摘要】：[目的]观察生理状态下人骨肉瘤细胞中p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38MAPKs)被磷酸化的过程。[方法]先进行ECFP-p38MAPK-Citrine(p38MAPK biosensor)融合蛋白表达载体的构建及鉴定,然后转染MG-63细胞24h后观察转染效率和融合蛋白表达情况。在荧光显微镜下,应用Meta Flour FRET 4.6软件测量转化生长因子-β1刺激MG-63细胞前后p38MAPK biosensor的荧光能量共振转移的变化情况。[结果]p38MAPK biosensor转染效率达30%~40%,均匀分布在胞质和胞核中。转化生长因子-β1刺激MG-63细胞后,胞质和胞核内荧光能量共振转移比值(Citrine/CFP)迅速增高,历时约30min达到最大值。特异性p38MAPK抑制剂SB-203580与细胞共孵育后,FRET比值逐渐减小。[结论]应用荧光能量共振转移技术使我们在活细胞生理状态下,实时动态监测p38MAPK被磷酸化的时空信息。
[Abstract]:[Objective] to observe the phosphorylation of p38 mitogen activated protein kinase (p38 mitogen-activated protein kinases) in human osteosarcoma cells under physiological condition. [Methods] we first constructed and identified the expression vector of ECFP-p38MAPK-Citrine (p38MAPK biosensor) fusion protein, then transfected MG-63 cell 24h, and observed the transfection efficiency and fusion protein expression. Under fluorescence microscope, Meta Flour FRET 4.6 software was used to measure the change of p38MAPK biosensor fluorescence energy resonance transfer in MG-63 cells stimulated by TGF - beta 1. [results the transfection efficiency of]p38MAPK biosensor was 30%~40% and distributed evenly in the cytoplasm and nucleus. After the TGF - beta 1 stimulated MG-63 cells, the fluorescence energy resonance transfer ratio (Citrine/CFP) in the cytoplasm and nucleus increased rapidly, and reached the maximum value of about 30min. After the specific p38MAPK inhibitor SB-203580 was incubated with the cells, the ratio of FRET decreased gradually. [Conclusion] using the fluorescence energy resonance technique, we can dynamically monitor the spatio-temporal information of phosphorylation of p38MAPK in live cell physiological state.
【作者单位】：新疆地方民族与地方病教育部重点实验室;石河子大学医学院第一附属医院检验科;石河子大学医学院第一附属医院骨一科;
【基金】：石河子大学课题(“TGFβ作用活细胞及p38MAPK信号通路的时空成像研究”,No.ZRKX2010ZD02-3)资助
【分类号】：R3416
【正文快照】： 0引言丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)是细胞内信号传导通路中重要的酶家族。这一家族包括了4个主要成员:c-jun氨基端激酶(JNK)、细胞外信号调节蛋白激酶(ERK1/2)、细胞外信号调节激酶(ERK5)和p38MAPK[1]。1993年Brewster[2]等首先发现p38MAPK是

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