Schubert Ralf, Geiger Helmut, Zielen Stefan, Baer Patrick C
Department of Pediatrics, Goethe University, Theodor Stern Kai 7, 60590 Frankfurt/M., Germany.
J Immunol Methods. 2009 Oct 31;350(1-2):200-4. doi: 10.1016/j.jim.2009.08.015. Epub 2009 Sep 3.
Despite expanded research in stem cell biology, little is known about the mechanisms underlying migration, growth, and differentiation of adipose-derived adult mesenchymal stem cells (ASC). The simultaneous measurement of intracellular pathways opens new avenues to gain further insights in these processes. We used the Cytometric Bead Array (CBA) Flex Set technology to simultaneously analyze protein phosphorylation after stimulation of ASC and compared the results with data generated by corresponding Western blots. Signal transduction of ASC was stimulated by epidermal growth factor (EGF) and analyzed by determining phosphorylation of mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK by Western blotting and CBA. After incubation with EGF, all MAPKs were significantly but differentially phosphorylated depending on time and dose. Furthermore, the ERK-response was abolished by EGF-R antagonist AG 1478 and kinase inhibitor PD98059, whereas p38 and JNK were only inhibited by AG1478. The stimulation and inhibition profiles between the two assays were highly comparable and the data were significantly correlated. In the present study we demonstrated that the CBA technology offers a reliable and convenient method for multiplexing of phospho-proteins in the evaluation of signal transduction pathways of adipose-derived mesenchymal stem cells.
尽管干细胞生物学研究不断扩展，但对于脂肪来源的成人间充质干细胞（ASC）迁移、生长和分化的潜在机制仍知之甚少。同时测量细胞内信号通路为深入了解这些过程开辟了新途径。我们使用细胞计数珠阵列（CBA）Flex Set技术，在刺激ASC后同时分析蛋白质磷酸化，并将结果与相应蛋白质印迹产生的数据进行比较。通过用表皮生长因子（EGF）刺激ASC的信号转导，并通过蛋白质印迹和CBA测定丝裂原活化蛋白激酶（MAPK）ERK、p38和JNK的磷酸化来进行分析。与EGF孵育后，所有MAPK均根据时间和剂量显著但有差异地磷酸化。此外，EGF-R拮抗剂AG 1478和激酶抑制剂PD98059消除了ERK反应，而p38和JNK仅被AG1478抑制。两种检测方法之间的刺激和抑制图谱具有高度可比性，且数据显著相关。在本研究中，我们证明CBA技术为评估脂肪来源间充质干细胞信号转导途径中的磷酸化蛋白多重检测提供了一种可靠且便捷的方法。
