血管内皮生长因子在鼠尾胶原凝胶诱导三维血管新生中的作用研究

背景:血管新生在组织工程研究中已引起了高度重视.血管内皮生长因子在二维平面培养中已证实能促进血管新生.目的:观察血管内皮生长因子在三维血管新生中的作用.方法:取SD大鼠骨髓,分离出内皮祖细胞.待细胞融合至70%～80%时添加鼠尾另一层胶原凝胶建立三维立体模型.实验组采用完全培养液含M199培养液、胎生血清加血管内皮生长因子及双抗;对照组培养液中不含血管内皮生长因子.培养第1,4,7,20天观察骨髓来源内皮祖细胞的体外培养和扩增情况并进行细胞鉴定.三维立体模型建立后第3,6,9,12天进行形态观察及定性定量分析.结果与结论:实验组内皮祖细胞在三维基质内向胶原基质内生长,24 h内即可出现向胶原内的出芽及浸润并逐渐形成分支样结构,对照组细胞生长慢,出芽慢,管状结构细小,向胶原内浸润的深度浅,网状结构稀疏,不完整.实验组新生血管数目显著高于对照组(P<0.01).取第3,6,9,12天的凝胶块检测,可见内皮素1、内皮型一氧化氮合成酶3表达阳性.结果表明,血管内皮生长因子能动员和诱导内皮祖细胞促进血管新生.鼠尾胶原凝胶可以诱导内皮祖细胞表现出血管新生中的迁移、增殖和发芽等步骤.

Vascular endothelial growth factor in three-dimensional angiogenesis induced by rat tail collagen gel

BACKGROUND: Angiogenesis attracts much attention in tissue engineering field. Previous research has proved that a two-dimensional culture of vascular endothelial growth factor (VEGF) promotes angiogenesis.OBJECTIVE: To evaluate the effect of VEGF on three-dimensional angiogenesis.METHODS: Endothelial progenitor cells were separated from the SD rat bone marrow. At about 70%-80% fusion, rat tail collagen gel was added to establish three-dimensional models. Samples in the experimental group were incubated in complete culture solution containing M199 culture media, fetal bovine serum, VEGF, and double antibody. The samples in the control group were incubated with VEGF-free culture media. In vitro culture and amplification of bone marrow-derived endothelial progenitor cells were determined at 1, 4, 7, and-20 days after incubation. Morphology and quantitative analysis were performed at 3, 6, 9, and 12 days after three-dimensional model establishment.RESULTS AND CONCLUSION: Endothelial progenitor cells grew from three-dimensional matrix into collagen matrix in the experimental group. Budding and infiltration were observed in the collagen within 24 hours, and branching-like structure was then gradually formed. Cells in the control group grew slowly, with slowing budding, small tubiform structure, superficial infiltration into COllagen, sparse network structure, and non-intact. Numbers of newborn vessels in the expedmental group were significantly greater than control group (P<0.01). A detection on gel block showed positive expressions of endothelin-1 and endothelial nitric oxide synthase-3 on the 3~(rd), 6~(th), 9~(th), and 12~(th) days. The results demonstrated that VEGF mobilized and induced endothelial progenitor cells in order to promote angiogenesis. Rat tail collagen gel induced endothelial progenitor cells which behaved migration, proliferation, and pullulation of angiogenesis.