骨髓基质细胞分泌蛋白的蛋白质质谱分析

目的通过Shotgun蛋白质组学分析,初步检测骨髓基质细胞(BMSCs)条件液中可能含有的蛋白质。方法将BMSCs条件液混匀后经超滤浓缩分为大于5kD和小于5kD两部分,并培养神经干细胞(NSCs),观察其后代中神经元、星形胶质细胞和少突胶质细胞的比例,鉴定出能调节NSCs分化的部分,并进行Shotgun蛋白质组学分析。结果BMSCs条件液分子量大于5kD的部分可以调节NSCs向神经元和少突胶质细胞方向分化,这部分条件液先经过聚丙烯酰胺凝胶电泳(SDS-PAGE)发现大部分蛋白集中在14kD以上,将蛋白条带酶解以后,经Shotgun分析共鉴定到456种蛋白质,其中154个相似蛋白质,17个假想蛋白质,56个未知蛋白质,剩余的229个蛋白质中大多为细胞骨架蛋白、分泌蛋白、信号转导蛋白、酶类和转运蛋白等。结论BMSCs分泌的多种蛋白质对NSCs的分化起调节作用,明确了BMSCs条件液中可能含有的成分。

Analysis of proteins secreted by bone marrow stromal cells using shotgun mass spectrometry

Objective Using shotgun mass spectrometry to detect proteins probably contained in bone marrow stromal cells(BMSCs) conditioned medium. Methods Mixed with BMSCs conditioned medium was divided into two parts which is(>5kD and <5kD) by means of ultrafiltration. The two parts were used to culture neural stem cells(NSCs) separately, and the proportions of neurons, astrocytes and oligodendrocytes in the offsprings of NSCs were calculated, then the effective part that could regulate the differentiation of NSCs was detected by Shotgun mass spectrometry. Results The BMSCs conditioned medium which is >5kD could promote the NSCs differentiate into more neurons and oligodendrocytes. The SDS-PAGE of this part showed that the most proteins were above 14kD, then the protein bands were enzymed. In total, 456 proteins were identified by Shotgun mass spectrometry after all the protein bands were enzymed, there were 154 similar proteins, 17 hypothetical proteins and 56 unknown proteins. And in the rest of 229 proteins, most of them were cytoskeletal proteins, secreted proteins, signal transduction proteins, enzymes, transporter and so on. Conclusion Many proteins secreted by BMSCs could regulate the differentiation of NSCs so as to prove the protein components probably existed in the BMSCs conditioned medium.