Background Bone marrow stromal cells protect hematopoietic cells and provide drug resistance by delivering bunch of variable proteins. Thus, alterations of protein expression are typically associated with cell-cell signal transduction and regulation of cellular functions. Methods Co-culture models of bone marrow stromal cells and hematopoietic cells are often used in studies of their crosstalk. Studies of altered protein expression initiated by stromal cell/hematopoietic cell interactions are an important new trend in microenvironmental research. There has been no report to date of global quantitative proteomics analysis of crosstalk between hematopoietic cells and stromal cells. In this study, we analyzed quantitative proteomes in a co-culture system of stromal HS5 cells and hematopoietic KG1a cells, and simultaneously tracked differentially expressed proteins in two types of cells before and after co-culture by stable isotope labeling by amino acids in cell culture (SILAC) method. Results We have shown that in co-cultured KG1a, 40 proteins (including CKAP4, LMNA, and SERPINB2) were upregulated and 64 proteins (including CD44, CD99, and NCAM1) were downregulated relative to KG1a alone. We utilized IPA analysis to discover that the NOD-like receptor signaling pathway was upregulated, whereas platelet activation was downregulated in co-cultured KG1a cells. Furthermore, 95 proteins (including LCP1, ARHGAP4, and UNCX) were upregulated and 209 proteins (including CAPG, FLNC, and MAP4) were downregulated in co-cultured HS5 relative to HS5 alone. The tight junction pathway was downregulated and glycolysis/gluconeogenesis pathway was dysfunctional in co-cultured HS5. Most importantly, the significantly differentially expressed proteins can also be confirmed using different co-cultured cell lines. Conclusion Altogether, we recommend such quantitative proteomics approach for the studies of the hematopoietic-stroma cross-talk, differentially expressed proteins and related signaling pathways identification. The differentially expressed proteins identified from this current SILAC method will provide a useful basis for ongoing studies of crosstalk between stromal cells and hematopoietic cells in co-culture systems. All these result suggested our ongoing studies can focus on the mechanisms underlying CKAP4 increase and CD44 decrease in co-cultured hematopoietic cells, and the increase of LCP1 and decrease of CAPG in co-cultured stromal cell. The proteomic profiles from the KG1a/stromal cell co-culture system give new molecular insights into the roles of these cells in MDS pathophysiology and related bone disease.

中文翻译：

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通过定量蛋白质组学 (SILAC) 方法从共培养的造血细胞和骨髓来源的基质细胞中筛选差异表达的蛋白质。

背景 骨髓基质细胞保护造血细胞并通过传递大量可变蛋白来提供耐药性。因此，蛋白质表达的改变通常与细胞间信号转导和细胞功能调节有关。方法 骨髓基质细胞和造血细胞的共培养模型常用于研究它们的串扰。由基质细胞/造血细胞相互作用引发的改变蛋白质表达的研究是微环境研究的重要新趋势。迄今为止，还没有关于造血细胞和基质细胞之间串扰的全球定量蛋白质组学分析的报告。在这项研究中，我们分析了基质 HS5 细胞和造血 KG1a 细胞共培养系统中的定量蛋白质组，并通过细胞培养中的氨基酸稳定同位素标记（SILAC）方法同时跟踪共培养前后两种细胞中差异表达的蛋白质。结果我们已经表明，在共培养的 KG1a 中，相对于单独的 KG1a，40 种蛋白质（包括 CKAP4、LMNA 和 SERPINB2）上调，64 种蛋白质（包括 CD44、CD99 和 NCAM1）下调。我们利用 IPA 分析发现 NOD 样受体信号通路上调，而血小板活化在共培养的 KG1a 细胞中下调。此外，与单独的 HS5 相比，在共培养的 HS5 中，95 种蛋白质（包括 LCP1、ARHGAP4 和 UNCX）上调，209 种蛋白质（包括 CAPG、FLNC 和 MAP4）下调。在共培养的 HS5 中，紧密连接通路被下调，糖酵解/糖异生通路功能失调。最重要的是，显着差异表达的蛋白质也可以使用不同的共培养细胞系来确认。结论 总之，我们推荐这种定量蛋白质组学方法用于造血-基质串扰、差异表达蛋白和相关信号通路鉴定的研究。从目前的 SILAC 方法中鉴定出的差异表达蛋白质将为正在进行的共培养系统中基质细胞和造血细胞之间的串扰研究提供有用的基础。所有这些结果表明，我们正在进行的研究可以集中在共同培养的造血细胞中 CKAP4 增加和 CD44 减少的机制上，和共培养的基质细胞中LCP1的增加和CAPG的减少。KG1a/基质细胞共培养系统的蛋白质组学特征为这些细胞在 MDS 病理生理学和相关骨病中的作用提供了新的分子见解。