促进内皮祖细胞生长的细胞外基质组织工程血管支架的研究

A novel scaffold for endothelial progenitor cells in tissue engineered vascular grafts

目的研究鼠骨髓来源内皮祖细胞（endothelial progenitor cells，EPCs）在不同类型细胞外基质支架上的生长特性，为EPCs生长寻找新的生物组织工程血管支架。方法EPCs种植于细胞外基质支架（extracellular matrix，ECM）上。培养不同时间点用免疫荧光技术鉴定并细胞记数；电镜观察支架表面结构及EPCs的生长情况；Western blotting法、real-time PCR法检测VWF（von Willebrand factor）蛋白及mRNA表达变化。结果在1、3、5h3个检测点的压缩组细胞贴壁率明显高于未压缩组（P〈0．01）。在1、3、7d压缩组的细胞数明显高于未压缩组（P〈0．05），10d后差异无统计学意义，且压缩组细胞形态较成熟，与内皮细胞相似，有一个较平整的细胞平面。Western blotting检测表明3、7、10dVWF蛋白在压缩支架上表达比未压缩支架上强，14d后两组表达相当。real-time PCR结果示3、7、10d压缩组VWF基因表达量明显高于未压缩组（P〈0．01），14d后两组表达无差异。结论压缩ECM更能促进EPCs黏附、增殖和分化，可作为一种新的合成人工血管的生物组织工程支架。

Objective To explore the characteristics of endothelial progenitor cells （EPCs） on different scaffolds and to find a new bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels. Methods EPCs were induced from mesenchymal stem cells isolated from rat bone marrow and seeded on ECM scaffold. The surface structure of the scaffold and gowth status of EPCs on the scaffold were observed and analysed by electron microscopy. The characteristics and number of those EPCs on different kinds of scaffolds were studied with EPC-specific VWF by immunofluorescence, Western blotting and real-time PCR technique at different time points. Results The cell adhesion rate at 1,3,5 h after seeded on pressed scaffold were higher than that on unpressed scaffolds （P 〈 0.01 ）. Pressed scaffolds has got a larger cell number （ P 〈 0. 05 ） at DIV1, DIV3, DIVT, but there was no significant difference after DIV10. Furthermore, cell shapes of EPCs on pressed scaffolds were more mature and more similar to endothelial cells. A level cell surface on pressed scaffolds was achieved. Western blotting assays revealed EPCs on pressed scaffolds expressed more protein VWF at DIV3, DIV7, DIV10. Real-time PCR results showed EPCs on the two different groups of scaffolds all expressed VWF gene, The quantity of their expression in the two groups were all enhanced after DIV7 （ P 〈 0. 05 ）. The quantity of VWF gene expression in the pressed group was much higher than that in the unpressed group at DIV3, DIV7, DIV10 （P 〈 0. 01 ）, but there was no significant difference after DIV14. Conclusions Pressed ECM scaffolds can promote adhesion, proliferation and differentiation on EPCs. Pressed scaffolds can be used as the matrix for EPC and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.