VEGF/PELA/bFGF混合微囊促进大鼠BMSCs成血管分化研究  

Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

作　　者：赵胜利[1] 殷杰 于庆贺 张国炜 闵少雄[1] ZHAO Shengli;YIN Jie;YU Qinghe;ZHANG Guowei;MIN Shaoxiong(Department of Spinal Surgery,Zhujiang Hospital of Southern Medical University,Guangzhou Guangdong,510282,P.R.China;Department of Hand Surgery,Ningbo No.6Hospital,Ningbo Zhejiang,315040,P.R.China)

机构地区：[1]南方医科大学珠江医院脊柱外科,广州510282 [2]宁波市第六医院手外科,浙江宁波315040

出　　处：《中国修复重建外科杂志》2019年第2期243-251,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：广东省自然科学基金资助项目(2014A030313348;2017A030313564);广州市科技计划项目资助项目(201607010266)~~

摘　　要：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。Objective To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycolpolylactic 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. Methods The 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. At21 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. Results The 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 coculture 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

关 键 词：BMSCS VEGF BFGF 聚乳酸-聚乙二醇-聚乳酸共聚物 内皮分化 

分 类 号：R68[医药卫生—骨科学]