Dahl Annika, Eriksson Peter S, Persson Anders I, Karlsson Gosta, Davidsson Pia, Ekman Rolf, Westman-Brinkmalm Ann
Institute of Clinical Neuroscience, Sahlgrenska University Hospital/Mölndal, Göteborg University, 431 80 Mölndal, Sweden.
Rapid Commun Mass Spectrom. 2003;17(19):2195-202. doi: 10.1002/rcm.1183.
It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo.
众所周知，神经干/祖细胞在体外的增殖和存活不仅依赖于外源性因子，还依赖于分泌到条件培养基中的自分泌因子。同样众所周知的是，鉴定分泌到条件培养基中的生物活性蛋白是一项重大挑战。最近，研究表明神经干/祖细胞会向条件培养基中分泌一种存活因子——胱抑素C。在此，我们展示了一种鉴定成年大鼠海马祖细胞培养的条件培养基中其他低分子量蛋白的方法。分析过程中采用了制备型二维凝胶电泳（2-DE）和质谱联用技术。我们能够鉴定出多种蛋白，包括Rho-鸟嘌呤核苷酸解离抑制剂1、磷脂酰乙醇胺结合蛋白（PEBP，也称为Raf-1激酶相互作用蛋白）、多聚泛素、亲免蛋白FK506结合蛋白12（FKBP12）以及胱抑素C。通过免疫方法证实了条件培养基中存在PEBP和FKBP12。所有巢蛋白阳性的祖细胞都对针对PEBP和FKBP12的抗体表现出免疫反应性。据我们所知，我们是首个使用这种制备型蛋白质组学方法在条件培养基中寻找干细胞因子的。该方法可用于鉴定体外干细胞/祖细胞分泌的新型生物活性蛋白。体外鉴定生物活性蛋白对于理解体内细胞的调控机制具有潜在重要意义。
