Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery
Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery
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DOI：
10.3791/55192
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发表时间：
2017-02-01
影响因子：
1.2
通讯作者：
Stoppe, Christian
中科院分区：
文献类型：
--
作者：
Emontzpohl, Christoph;Simons, David;Stoppe, Christian
内皮祖细胞（Endothelial progenitor cells，EPCs）是在缺氧等病理条件下从骨髓中募集的细胞，在缺血组织的新生血管形成中起关键作用。EPCs的起源、分类和表征是复杂的;尽管如此，已经建立了EPCs的两种突出的亚型：所谓的“早期”EPCs（随后称为早期EPCs）和晚期生长EPCs（晚期EPCs）。它们可以通过生物学特性以及它们在体外培养期间的外观进行分类。虽然“早期”EPCs在外周血来源的单核细胞在EC特异性培养基中培养后不到一周内出现，但可以在2-3周后发现晚期生长的EPCs。晚期生长的EPCs被认为直接参与新生血管形成，主要是通过其分化为成熟内皮细胞的能力，而“早期”EPCs表达各种血管生成因子作为内源性货物，以旁分泌方式促进血管生成。在心肌缺血/再灌注（I/R）期间，多种因素控制EPCs归巢至血管形成区域。巨噬细胞迁移抑制因子（MIF）是一种趋化因子样促炎性和普遍表达的细胞因子，并且最近被描述为在生理浓度下作为EPCs迁移的关键调节剂（1）。有意思的是，MIF储存在细胞内池中，并可在几次刺激后迅速释放到血流中该方案描述了基于CD 34阳性选择从成人外周血可靠分离和培养早期EPC的方法，随后在含有内皮生长因子的培养基中在纤连蛋白上培养。用于针对心脏手术患者血清样品的体外迁移测定的包被板。此外，迁移的影响，MIF对EPCs的趋化性相比，其他众所周知的血管生成刺激细胞因子的证明。
Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called "early" EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While "early" EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas "early" EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations(1). Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction).This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other wellknown angiogenesis-stimulating cytokines is demonstrated.