兔外周血内皮祖细胞种植支架的实验研究

Experimental study on rabbit endothelial progenitor cells-seeded stents in vitro

目的体外制备兔内皮祖细胞(EPC)种植支架,并评价其可行性。方法采用密度梯度离心法分离兔外周血单个核细胞,接种于人纤维连接蛋白包被的培养板上,予含内皮细胞生长添加剂30μg/mL的M199培养基培养。培养2周后进行免疫荧光染色和免疫细胞化学法鉴定EPC,检测其增殖、迁移、黏附和分泌血管内皮生长因子(VEGF)、粒细胞集落刺激因子(G-CSF)和一氧化氮(NO)的能力。培养的EPC消化后,将其浇注到有人纤维连接蛋白包被和无包被的裸支架上,6 d后行扫描电镜和荧光显微镜检查,观察其表面细胞的生长情况。结果单个核细胞培养14 d后,出现大量梭形细胞,免疫荧光染色显示细胞可同时摄取Dil标记的乙酰化低密度脂蛋白和结合FITC标记的凝集素者,表明为正在分化的EPC,同时证实其表达内皮细胞的特异标志vWF。培养2周后的EPC数量呈指数增加;EPC的黏附率为96.7%±3.2%;迁移为15±4细胞/×400视野;可分泌VEGF、G-CSF和NO。与无包被的裸支架组相比,有纤维连接蛋白包被的支架组,EPC黏附多(27.80±4.26细胞/×400视野比6.10±3.07细胞/×400视野,P<0.05),内皮化完全。结论EPC可向内皮细胞方向分化,并且具有高增殖、高迁移和高黏附的能力。采用EPC在体外制备细胞种植支架是可行的,而纤维连接蛋白可加速其黏附。

Objective To fabricate rabbit endothelial progenitor cells（EPCs）-seeded stents in vitro and evaluate its feasibility. Methods The mononuclear cells of rabbit were separated by density-gradient eentrifugation using lymphocyte isolation. Cells were seeded on fibronectin-coated 6-well plates and maintained on M199 with endothelial cell growth supplements （ECGS, 30 μg/mL）. After 2 weeks, cultured cells were identified by immunofluorescence and immunocytochemistry. EPCs proliferation and migration were measured by drawing a growth curve and modified Boyden chamber assay. EPCs adhesion assay was performed by replating EPCs on fibronectin-coated dishes. VEGF and G-CSF were assayed by ELISA while NO was tested by nitrate reductase method. EPCs were poured on fibronectin-coated or uncoated stents. After 6 days, scanning electron microscopy and fluorescence microscopy observation were performed. Results About 14 days after seeding, a majority of spindle-like cells appeared which were characterized as cells, double positive for Dil-acLDL and FITC-UEA-I. EPCs were further confirmed by its expression of the endothelial cell marks of vWF. After 2 weeks culture, EPCs proliferated in exponential growth. They showed high ability of adhesion （ 96. 7 ± 3.2% ） , migration （ 15 ± 4 ） and could secret VEGF, G-CSF and NO. Compared with uncoated stents group, more EPCs migrated and adhered on fibroneetin-coated stents group （27. 80 ± 4. 26 vs 6. 10 ± 3.07cells/x 400 field, P 〈 0. 05 ）. Endothelialization of coated stents was visible under the scanning electron microscopy. Conclusion EPCs can differentiate to endothelial lineage and posses high ability of proliferation, migration and adhesion. It is feasible to fabricate rabbit EPCs-seeded stents in vitro. Stents coated with fibronectin are easier for cells to adhere on.