共价结合血管内皮生长因子促进内皮祖细胞在去细胞瓣上的粘附和增殖  

PEG-mediated covalent binding of VEGF to decellularized aortic valves promotes adhesion and proliferation of endothelial progenitor cells

作　　者：周建良[1] 邹明晖[1] 陈义初[1] 卢翠芬[2] 史嘉玮[1] 董念国[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心血管外科,湖北武汉430022 [2]湖北大学化学化工学院,湖北武汉430062

出　　处：《南方医科大学学报》2011年第9期1474-1479,共6页Journal of Southern Medical University

基　　金：国家"863"高技术研究发展计划(2009AA03Z420);国家自然科学基金(30872540)~~

摘　　要：目的对去细胞主动脉瓣进行聚乙二醇(PEG)交联和血管内皮生长因子(VEGF)共价结合,以改善去细胞主动脉瓣生物学性能。方法枝化状聚乙二醇末端的丙烯酰基与引入到去细胞主动脉瓣上的巯基,通过迈克尔加成反应完成对去细胞主动脉瓣的PEG交联;通过PEG交联去细胞主动脉瓣上的未饱和丙烯酰基与VEGF中半胱氨酸残基上的巯基发生另一个迈克尔加成反应,对去细胞主动脉瓣进行VEGF共价结合,酶联免疫吸附试验和免疫荧光测定VEGF修饰效果;在去细胞主动脉瓣、PEG交联去细胞主动脉瓣和VEGF共价结合的PEG交联去细胞主动脉瓣上种植内皮祖细胞,培养10 d后行瓣膜上DNA含量测定、苏木素-伊红染色和扫描电镜。结果免疫荧光和ELISA检测显示:每片PEG交联去细胞主动脉瓣最大结合(908.94±0.27)pg VEGF;与去细胞主动脉瓣和PEG交联去细胞主动脉瓣相比,VEGF共价结合的PEG交联去细胞主动脉瓣明显促进内皮祖细胞的黏附和增殖,并且可在瓣膜表面形成一层连续的单细胞层。结论借助PEG,VEGF能有效地共价结合去细胞主动脉瓣,促进细胞的粘附和增殖,有利于组织工程心脏瓣膜的构建。Objective To improve the biological properties of decellularized aortic valves by polyethylene glycol（PEG）-mediated covalent incorporation of vascular endothelial growth factor（VEGF）.Methods PEG crosslinking of decellularized aortic valves were completed via a Michael-type addition reaction,followed by covalent incorporation of VEGF through another Michael-type addition reaction between the unsaturated propylene acyl of PEG and the thiol groups on cysteine residues of VEGF.The effect of VEGF incorporation was evaluated by enzyme-linked immunosorbent assay（ELISA） and immune fluorescence assay.The endothelial progenitor cells（EPCs） were seeded on decellularized aortic valves with or without these modifications,and after 10 days of culture,the valves were examined for DNA content and by hematoxylin-eosin staining and scanning electron microscopy.Results Immune fluorescence and ELISA showed that the maximal VEGF incorporation on the decellularized aortic valve reached 908.94±0.27 pg.Compared with the unmodified valves and the valves with PEG crosslinking,decellularized aortic valves with covalent incorporation of VEGF significantly promoted the adhesion and proliferation of EPCs,which formed a confluent cell monolayer on the valve surface.Conclusions PEG-mediated covalent incorporation of VEGF in the decellularized aortic valves improves the adhesion and proliferation of the seeded EPCs to facilitate the construction of tissue-engineered heart valves.

关 键 词：去细胞主动脉瓣 聚乙二醇 迈克尔加成反应 血管内皮生长因子 内皮祖细胞 

分 类 号：R329.26[医药卫生—人体解剖和组织胚胎学]