摘要
本文旨在探讨不同细胞外基质(extracellular matrix,ECM)对大鼠骨髓来源的晚期内皮祖细胞(endothelial progenitorcells,EPCs)生物学特性的影响。密度梯度离心法分离大鼠骨髓单核细胞,应用M199完全培养基进行体外培养,培养基中添加15%胎牛血清、10μg/L血管内皮生长因子(vascular endothelial growth factor,VEGF)及5μg/L碱性成纤维细胞生长因子(basicfibroblast growth factor,bFGF)。在接种细胞前,培养板分别用纤维粘连蛋白(fibronectin,Fn)、层粘连蛋白(laminin,Ln)或鼠尾胶原(collagen,Col)包被,定义为Fn组、Ln组及Col组。传至第三代EPCs(晚期EPCs)时,采用CCK-8法、EdU标记法、粘附能力测定实验、划痕实验及Matrigel法分别检测细胞增殖、粘附、迁移及血管形成情况;应用荧光定量RT-PCR检测内皮分化标志基因vWF和CD31的表达;应用流式细胞术检测细胞凋亡。结果显示,Fn组和Col组细胞增殖能力强于Ln组;Fn组细胞粘附能力强于Col组和Ln组;Fn组和Col组细胞迁移能力强于Ln组(P<0.05);Fn组血管形成能力强于Col组及Ln组(P<0.05);Ln组血清饥饿所诱导的细胞凋亡率大于Fn组和Col组(P<0.01),但三组vWF和CD31基因表达及自发凋亡之间均无明显差异。以上结果表明,EPCs的生物学特性受ECM的影响,这将为人工心、血管的ECM血管支架材料选择方面提供一定的参考依据。
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 M 199 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 prolif- eration ability of Fn and Col groups were higher than that of Ln group; Fn group showed increased adhesion compared to Col and Lngroups （P 〈 0.01）; The migration ability of Fn and Col groups were higher than that of Ln group. Moreover, Fn group showed in- creased 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 v WF 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.
出处
《生理学报》
CAS
CSCD
北大核心
2013年第4期409-416,共8页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China(No.30900290
31270993)
Program for New Century Excellent Talents in Universities of Ministry of Education
China(No.NCET-10-0922)
the Natural Science Foundation of Shandong Province
China(No.ZR2009CQ027
ZR2011CQ030)
the Medicine and Health Science and Technology Project of Shandong Province
China(No.2011QZ024)
