Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method
Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method
复制标题
使用 SILAC 方法对两种骨髓基质细胞系进行定量蛋白质组分析和比较。
DOI：
10.1016/j.exphem.2016.08.002
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发表时间：
2016-11-01
影响因子：
2.6
通讯作者：
Han, Xiaofeng
中科院分区：
文献类型：
--
作者：
Li, Xiang;Wan, Ting;Han, Xiaofeng
与骨髓细胞共培养的两种人骨髓基质细胞系HS 5和HS 27 a经常用于骨髓微环境中的细胞与造血细胞之间的串扰研究。蛋白质表达的改变通常与细胞信号转导和细胞功能的调节相关。许多研究集中在细胞共培养模型中导致功能差异的关键蛋白质，但尚未进行HS 5和HS 27 a的全球定量蛋白质组分析。我们采用细胞培养中氨基酸稳定同位素标记（SILAC）方法，使用精氨酸和赖氨酸两种稳定同位素分别标记两种细胞系中的蛋白质。标记的蛋白质通过2-D超高分辨率液相色谱-LTQ/Orbitrap质谱分析。在细胞系中的4，213个独特的鉴定和注释蛋白质中，在两个独立的实验中检测到1，462个。其中，相对于HS 5细胞，69个在HS 27 a中表现出显著的上调和48个显著的下调（>95%置信度）。基因本体论术语和通路分析表明，差异调节蛋白参与细胞运动、细胞间信号传导和相互作用以及血液系统发育和功能。在基因组和蛋白质组数据库中共鉴定出55个项目。对从两个数据库中鉴定的28个差异表达蛋白中随机选择的7个蛋白进行定量逆转录聚合酶链反应和Western印迹，并参与顶部网络/途径。我们观察到当整合素α V在HS 27 a细胞中被抑制时，共培养的KG 1a细胞中的凋亡减少，这表明整合素α V在共培养系统中的功能作用。本文所述的整合基因组/蛋白质组学方法和所鉴定的蛋白质将为进一步阐明骨髓微环境中的分子机制和正在进行的共培养系统中基质细胞和骨髓瘤细胞之间的串扰研究提供有用的基础。版权所有（C）2016 ISEH -国际实验血液学学会。爱思唯尔公司出版
Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow micro-environment and hematopoietic cells. Altered expression of proteins is typically associated with cell cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography-LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin alpha V was inhibited in HS27a cells, which suggested the functional role of integrin alpha V the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems. Copyright (C) 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.