Lee Sang Kwang, Kim Yongtae, Kim Sung-Soo, Lee Jeong Hwa, Cho Kun, Lee Sang Sook, Lee Zee-Won, Kwon Kyung-Hoon, Kim Young Hye, Suh-Kim Haeyoung, Yoo Jong Shin, Park Young Mok
Mass Spectrometry Research Center, Korea Basic Science Institute, Daejeon, Republic of Korea.
Proteomics. 2009 Sep;9(18):4389-405. doi: 10.1002/pmic.200900165.
Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.
间充质干细胞(MSCs)是多能细胞,具有分化为各种间充质组织的能力,如骨、软骨、脂肪、肌腱、肌肉和骨髓基质。然而,经过几次传代后,它们会失去多谱系分化的能力。已知碱性成纤维细胞生长因子(bFGF)可提高MSCs在体外的生长速率、分化潜能和形态变化。在本报告中,我们使用二维电泳(2-DE)结合质谱(MS)来鉴定在含bFGF培养基中生长的MSCs细胞膜水平上差异表达的蛋白质。通过生物素-抗生物素蛋白亲和柱分离的细胞表面蛋白在三次重复实验中通过2-DE进行分离。通过四极杆飞行时间串联质谱鉴定出总共15种差异表达的蛋白质。在含bFGF培养基培养的MSCs中,9种蛋白质上调,6种蛋白质下调。通过蛋白质免疫印迹分析证实了三种肌动蛋白相关蛋白,即F-肌动蛋白封端蛋白亚基α-1、肌动蛋白相关蛋白2/3复合物亚基2和肌球蛋白调节轻链2的表达水平。结果表明,F-肌动蛋白封端蛋白亚基α-1、肌动蛋白相关蛋白2/3复合物亚基2和肌球蛋白调节轻链2的表达水平在bFGF诱导的MSCs形态变化中很重要。