Sun Jin-Long, Zhang Xiao-Yun, Cui Xiao-Dong, Lu Hong-Ying, Yin Qing-Ling, Jing Xu, Wu Hai-Yan, Cheng Min
Department of Physiology, Weifang Medical University, Weifang 261053, China. E-mail:
Sheng Li Xue Bao. 2013 Aug 25;65(4):409-16.
The present study was designed to investigate the effects of various extracellular matrix (ECM) proteins on the biological characteristics of late endothelial progenitor cells (EPCs). Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in complete M199 medium, which contained 15% fetal calf serum, 10 μg/L vascular endothelial growth factor (VEGF) and 5 μg/L basic fibroblast growth factor (bFGF). EPCs were plated on substrates containing fibronectin (Fn), laminin (Ln) or rat tail tendon collagen (Col), and the corresponding cells were defined as Fn, Ln and Col groups. The 3rd generation EPCs, namely late EPCs, were harvested. The proliferation, adhesion, migration and the ability of forming tubes were assayed using CCK-8, adhesion test, wound healing assay and Matrigel, respectively. The mRNA expressions of endothelial cell differentiation markers, vWF and CD31, were analyzed by real time RT-PCR. The apoptosis was assayed by flow cytometry (FCM). The results showed that cell proliferation ability of Fn and Col groups were higher than that of Ln group; Fn group showed increased adhesion compared to Col and Ln groups (P < 0.01); The migration ability of Fn and Col groups were higher than that of Ln group. Moreover, Fn group showed increased tube formation abilities compared to Col and Ln groups (P < 0.05). Although 24-hour free-serum-induced apoptosis in Ln group was the highest, there was no difference of auto-apoptosis among the three groups. Furthermore, the mRNA expressions of vWF and CD31 exhibited no difference among the three groups. These results suggest the ECM affects the biological functions of late EPCs, which would have a high probability of providing new directions that lead to the development of artificial heart and blood vessels.
本研究旨在探讨各种细胞外基质（ECM）蛋白对晚期内皮祖细胞（EPCs）生物学特性的影响。采用密度梯度离心法分离大鼠骨髓单个核细胞，将其培养于完全M199培养基中，该培养基含有15%胎牛血清、10 μg/L血管内皮生长因子（VEGF）和5 μg/L碱性成纤维细胞生长因子（bFGF）。将EPCs接种于含有纤连蛋白（Fn）、层粘连蛋白（Ln）或大鼠尾腱胶原（Col）的基质上，相应细胞分别定义为Fn组、Ln组和Col组。收获第3代EPCs，即晚期EPCs。分别采用CCK-8法、黏附试验、划痕试验和基质胶检测细胞的增殖、黏附、迁移及成管能力。通过实时RT-PCR分析内皮细胞分化标志物vWF和CD31的mRNA表达。采用流式细胞术（FCM）检测细胞凋亡情况。结果显示，Fn组和Col组的细胞增殖能力高于Ln组；与Col组和Ln组相比，Fn组的黏附能力增强（P < 0.01）；Fn组和Col组的迁移能力高于Ln组。此外，与Col组和Ln组相比，Fn组的成管能力增强（P < 0.05）。虽然Ln组24小时无血清诱导凋亡率最高，但三组细胞的自发凋亡率无差异。此外，三组vWF和CD31的mRNA表达无差异。这些结果表明，ECM影响晚期EPCs的生物学功能，这很有可能为人工心脏和血管的开发提供新的方向。
