Identification of regulatory Hck and PAI-2 proteins in the monocyte response to PEG-containing matrices.
Identification of regulatory Hck and PAI-2 proteins in the monocyte response to PEG-containing matrices.
复制标题
鉴定单核细胞对含 PEG 基质的反应中的调节性 Hck 和 PAI-2 蛋白。
DOI：
10.1016/j.biomaterials.2009.04.007
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发表时间：
2009
期刊：
影响因子：
14
通讯作者：
Kao,WeiyuanJ
中科院分区：
文献类型：
--
作者：
Zuckerman,SeanT;Brown,JamesF;Kao,WeiyuanJ
质谱分析是一种强大的蛋白质组学工具，使研究人员能够调查细胞的整体蛋白质组。这项技术最近才被用来研究细胞与材料的相互作用。我们之前已经发现材料稀缺和贴壁细胞有限是细胞与材料相互作用的质谱分析面临的挑战。粘附于组织培养聚（苯乙烯）的 U937 用作识别粘附单核细胞表达的蛋白质的模型系统，并通过离线耦合 MALDI-ToF/ToF (LC-MALDI) 的 HPLC 进行分析。我们从粗制 U937 单核细胞裂解物的两个阳离子级分中鉴定出了 645 种蛋白质。根据与单核细胞物质炎症和伤口愈合相关的文献检索，从 645 种蛋白质中选择了 43 种感兴趣的蛋白质。 40S 核糖体蛋白 S19 和酪氨酰 tRNA 合成酶等蛋白质凸显了 LC-MALDI 识别与单核细胞-物质相互作用相关的蛋白质（目前尚未探索）的能力。我们使用基于 PEG 的半互穿聚合物网络和纯 PEG 水凝胶，分别使用吡唑并嘧啶小分子抑制剂 PP2 和外源性尿激酶纤溶酶原激活剂添加物，研究对 Src 家族激酶 Hck 和纤溶酶原激活剂抑制剂 2 (PAI-2) 的表面依赖性影响。 Hck 在细胞粘附方面已得到充分研究，而 PAI-2 在细胞与材料相互作用方面几乎未知。 TCPS 和纯 PEG 水凝胶上的 U937 分泌相似水平的炎症细胞因子和明胶酶 MMP-9。仅 PEG 水凝胶上单核细胞的 MCP-1 分泌与 Hck 无关，而 TCPS 上的 U937 中的 MCP-1 分泌则依赖于 Hck。总体而言，粘附于 sIPN 的 U937 分泌低水平的可溶性明胶酶 MMP-9、IL-1β、TNF-α、IL-6 和 MCP-1，与 Hck 和 PAI-2 无关。这项工作表明，与 TCPS 相比，粘附于 PEG 材料的单核细胞的表面依赖性蛋白质表达发生了显着变化。
Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. This technique has only recently been employed to investigate cell–material interactions. We had previously identified material scarcity and limited adherent cells as challenges facing mass spectrometric analysis of cell–material interactions. U937 adherent to tissue culture poly(styrene) was used as a model system for identifying proteins expressed by adherent monocytes and analyzed by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate. Forty three proteins of interest from the 645 were chosen based on literature searches for relevance to monocyte–material inflammation and wound healing. Proteins such as 40S ribosomal protein S19 and tyrosyl tRNA synthetase highlight the ability of LC-MALDI to identify proteins relevant to monocyte–material interactions that are currently unexplored. We used PEG-based semi-interpenetrating polymer networks and PEG-only hydrogels to investigate surface dependent effects on the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2) using the pyrazolo pyrimidine small molecule inhibitor PP2 and exogenous urokinase plasminogen activator addition, respectively. Hck is well researched in cell adhesion while PAI-2 is virtually unknown in cell–material interactions. U937 on TCPS and PEG-only hydrogels secreted similar levels of inflammatory cytokines and gelatinase MMP-9. MCP-1 secretion from monocytes on PEG-only hydrogels was Hck independent in contrast to Hck-dependent MCP-1 secretion in U937 on TCPS. Overall, U937 adherent to sIPNs secrete low levels of soluble gelatinase MMP-9, IL-1β, TNF-α, IL-6, and MCP-1 independent of Hck and PAI-2. This work demonstrates significant changes in surface dependent expression of proteins from monocytes adherent to PEG-based materials compared to TCPS.