中国修复重建外科杂志

中国修复重建外科杂志

VEGF/PELA/bFGF 混合微囊促进大鼠 BMSCs 成血管分化研究

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目的观察 VEGF/聚乳酸-聚乙二醇-聚乳酸共聚物(polylactide-polyethyleneglycol-polylactic acid copolymer,PELA)/ bFGF 对 SD 大鼠 BMSCs 成血管分化作用。 方法经全骨髓贴壁法分离培养 BMSCs 并传代。取第 3 代 BMSCs 行 Wright-Giemsa 染色及流式细胞术鉴定。利用复乳溶剂挥发法制备 VEGF/PELA/bFGF(A 组)、PELA/bFGF(B 组)、VEGF/PELA(C 组)及 PELA(D 组)微囊。对 PELA 微囊行降解性能及细胞毒性检测,VEGF/PELA/bFGF 混合微囊行 VEGF 及 bFGF 体外释放检测。取 4 组微囊缓释上清液分别与第 3 代 BMSCs 培养。培养 1、3、7、14、20 d 于倒置显微镜下观察细胞形态变化;21 d 时,行摄取 Dil 标记的乙酰化低密度脂蛋白(DiI-labeled acetylated low density lipoprotein,Dil-ac-LDL)及 FITC 标记的荆豆凝集素(FITC-labeled ulex europaeus agglutinin I,FITC-UEA-I)实验,观察细胞摄取能力;同时行 Matrigel 基质胶小管形成实验,定量分析小管形成指标(节点数、交叉点数、主干数及主干长度)。 结果经鉴定分离培养的细胞为 BMSCs。PELA 微囊在 37℃ 恒温条件下降解时程超过 20 d,且对 BMSCs 活力无明显影响。VEGF/PELA/bFGF 混合微囊体外释放检测显示,至 20 d 时 VEGF、bFGF 累积释放量分别超过 95%、80%。倒置显微镜下观察共培养期间 A、B、C 组 BMSCs 均出现形态学变化,其中 A、B 组 20 d 细胞呈典型内皮细胞“铺路石”样改变。荧光显微镜观察,A 组双荧光标记细胞最多,B 组次之,C 组较少,D 组几乎不具备内皮细胞特有的摄取功能。Matrigel 基质胶小管形成实验示,A、B 组诱导细胞在 Matrigel 基质胶上均形成网格状结构;定量分析显示 A、B 组节点数、交叉点数、主干数以及主干长度均多于 C、D 组(P<0.05)。A 组节点数以及主干长度多于 B 组(P<0.05);两组交叉点数、主干数差异无统计学意义(P>0.05)。 结论VEGF/PELA/bFGF 混合微囊具有显著促进 BMSCs 成血管分化的能力。

ObjectiveTo observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. MethodsThe BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined, and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. ResultsThe isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant (P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B (P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B (P>0.05). ConclusionVEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

关键词: BMSCs; VEGF; bFGF; 聚乳酸-聚乙二醇-聚乳酸共聚物; 内皮分化

Key words: Bone marrow mesenchymal stem cells; vascular endothelial growth factor; basic fibroblast growth factor; polylactide-polyethyleneglycol-polylactic acid copolymer; endothelial differentiation

引用本文: 赵胜利, 殷杰, 于庆贺, 张国炜, 闵少雄. VEGF/PELA/bFGF 混合微囊促进大鼠 BMSCs 成血管分化研究. 中国修复重建外科杂志, 2019, 33(2): 243-251. doi: 10.7507/1002-1892.201808099 复制

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