体外构建经血管内皮生长因子共价修饰去细胞瓣的研究

Establishment of PEGylated decellularized valves modified by vascular endothelial growth factor in vitro

目的基于迈克尔加成反应，体外构建经血管内皮生长因子（VEGF）共价修饰的去细胞猪主动脉瓣（DPAV）复合材料，探讨复合瓣膜的力学性能和生物学性能。方法应用枝状化聚乙二醇（PEG）对去细胞的猪主动脉瓣PEG化，PEG化去细胞瓣（PEG-DPAV）与VEGF上的巯基发生反应，将VEGF共价修饰到去细胞瓣上（VEGF-PEG-DPAV）。检测VEGF共价修饰去细胞瓣的效果；对复合瓣膜在周向和径向上力学性能进行测试；通过差示扫描量热仪（DSC）、溶血实验和细胞毒性实验测试复合瓣膜的生物学性能。结果通过红外光谱和免疫荧光检测证实VEGF成功共价修饰到去细胞瓣上，苏木素．伊红（HE）染色和扫描电镜显示去细胞瓣膜交联PEG后，纤维增粗、排列整齐，结构更加致密。力学性能测试显示去细胞瓣PEG化后可以改善部分力学性能。DSC显示DPAV、PEG．DPAV和VEGF-PEG-DPAV组瓣膜的变性温度分别为（65．64±0．15）℃、（74．94±0．10）℃和（74．88±0．05）℃。溶血实验显示DPAV、PEG-DPAV和VEGF-PEG-DPAV组溶血率分别为（0．23±0．01）％、（0．24±0．01）％和（0．23±0．01）％。DPAV、PEG-DPAV和VEGF-PEG-DPAV组瓣膜均未表现出细胞毒性。结论成功制备经VEGF修饰的PEG化去细胞瓣，为体外去细胞瓣的改性研究提供-种可行性方法。

Objective We established decellularized valves modified by vascular endothelial growth factor （VEGF） in vitro based on michael addition reaction, and explored their mechanical and bio- logical properties. Methods 4 - arm - polyethylene glycol （PEG） - acrylate was used to PEGylate the de- cellularized aortic valves. The PEGylated decellularized valves were modified by VEGF in vitro. The estab- lishment result of composite valves was observed. Mechanical properties of valves were tested at circumfer- ential and radial directions. Their biological properties were measured by DSC, hemolytic and cytotoxicity test. Results We confirmed that PEGylated decellularized valves were successfully modified by VEGF via Fourier transform infrared spectroscopy and immunofluorescenee. The decellularized results showed colla- gen fibers thickened in alignment. Mechanics Performance Testing showed that PEGylated valves could im- prove part mechanical properties. DSC displayed denaturation temperatures of DPAV, PEG - DPAV and VEGF - PEG - DPAV were （65.64 ±0. 15 ） ℃, （74. 94 ± 0. 10） ℃ and （74. 88 ± 0. 05 ） ℃ respective- ly. Hemolytic test showed hemolysis ratios were （ 0. 23 ± 0. 01 ） % , （ 0. 24 ± 0. 01 ） % and （ 0. 23 ± 0. 01 ） % , respectively. No cytotoxicity was observed in DPAV, PEG - DPAV and VEGF - PEG - DPAV. Conclusion We successfully established PEGylated deeellularized valves modified by VEGF, which pro- vided a feasible method in scaffold modification of decellularized valves in vitro.