Skalnikova Helena, Halada Petr, Vodicka Petr, Motlik Jan, Rehulka Pavel, Hørning Ole, Chmelik Josef, Nørregaard Jensen Ole, Kovarova Hana
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic.
Proteomics. 2007 Jun;7(11):1825-38. doi: 10.1002/pmic.200600867.
The mechanisms that regulate the maintenance of stem cell self-renewal versus differentiation are complex and remain mostly unknown. Understanding neurogenesis and neural cell differentiation presents a unique challenge for the treatment of nervous system disorders. To gain more insight into molecular mechanisms of the differentiation of neural cells, we combined the advantage of porcine fetal neural stem cells (NSCs) in vitro differentiation model and proteomic analysis. Using 2-DE followed by MS, we profiled constituent proteins of NSCs and their differentiated progenies at first and then indicated protein species that were significantly up- or down-regulated during the differentiation. The largest identified group of constituent proteins was related to RNA and protein metabolism and processing, including chaperones, and the second largest consisted of proteins involved in cell organization (cytoskeleton and annexins). Differentiation of neural cells was found to be accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage, and redox regulation. Additional immunoblot analysis verified the induction of alpha-B crystallin and heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1. Furthermore, immunocytochemistry demonstrated specific localization of alpha-B crystallin in the cytoplasm or nucleus of glial cells and confirmed cellular expression patterns of hnRNPs A1 and A2/B1. These findings represent a significant step towards understanding neural cell differentiation and identification of the regulatory proteins associated with this process.
调控干细胞自我更新与分化维持的机制十分复杂，且大多仍不为人知。了解神经发生和神经细胞分化对于治疗神经系统疾病而言是一项独特的挑战。为了更深入地洞察神经细胞分化的分子机制，我们结合了猪胎儿神经干细胞（NSCs）体外分化模型的优势和蛋白质组学分析方法。首先利用双向电泳（2-DE）随后进行质谱分析（MS），我们对神经干细胞及其分化后代的组成蛋白进行了分析，然后指出了在分化过程中显著上调或下调的蛋白质种类。鉴定出的最大一组组成蛋白与RNA和蛋白质代谢及加工有关，包括分子伴侣，第二大组由参与细胞组织（细胞骨架和膜联蛋白）的蛋白质组成。研究发现神经细胞分化伴随着与DNA和RNA结合、mRNA加工与转运、应激反应、铁储存及氧化还原调节相关的蛋白质表达变化。额外的免疫印迹分析证实了α-B晶状体蛋白以及不均一核核糖核蛋白（hnRNPs）A1和A2/B1的诱导表达。此外，免疫细胞化学显示α-B晶状体蛋白在神经胶质细胞的细胞质或细胞核中具有特异性定位，并证实了hnRNPs A1和A2/B1的细胞表达模式。这些发现代表了在理解神经细胞分化以及鉴定与该过程相关的调控蛋白方面迈出的重要一步。
