枝状化聚乙二醇修饰的去细胞猪主动脉瓣膜内皮化的性能研究

The endothelialization properties of decellularized porcine aortic valve modified with branched polyethylene glycol

目的观察枝状化聚乙二醇（PEG）修饰后的去细胞猪主动脉瓣（DPAV）的内皮细胞生长性能。方法自合成枝状化聚乙二醇（4-arm—PEG—acrylate），采用核磁共振氢谱（1HNMR），对复合物进行表征检测，利用4-arm。PEG．amylate上的丙烯酸酯与巯基化的DPAV结合对其进行PEG化，体外获取人脐静脉内皮细胞，以细胞密度为1X10。接种于各组瓣膜支架材料上，8d后MTS试剂、苏木素-伊红（HE）染色、扫描电镜（SEM）评价内皮细胞的生长。结果。HNMR（D2O）：3．51～3．86ppm处PEG信号峰，4．14（4H，CH，OAr），5．80～6．48ppm处丙烯酸酯双键。SEM可见枝状化㈨（；修饰的DPAV胶原纤维交联增粗，人脐静脉内皮细胞在DPAV上种植8天DPAV组细胞数为（0．88±0．03）×105／cm，PEG—DPAV组细胞数为（1．18±0．04）×105／cm2，PEG—DPAV组高于DPAV组（P〈0．05）。HE染色和SEM可见两组瓣膜支架表面仅有散在的细胞存在，呈梭形或不规则形，细胞数PEG—DPAV高于DPAV组，部分可见细胞问连接紧密形成一层近似融合的细胞层，但细胞紧惭连接不连续。结论枝状化PEG修饰的去细胞猪主动脉瓣膜能够提高内皮细胞的黏附增殖，但卸不能在去细胞瓣膜支架表面形成一层完整的融合层，因此PEG化的瓣膜并不能达到理想的内皮化，有必要进行进一步修饰。

Objective To investigate the endothelialization performance of decellularized porcine aortic valve （DPAV） modified with branched polyethylene glycol. Methods Synthesis of 4 -arm- Poly- etbyl glycol （PEG） - acrylate, characterized by 1H nuclear magnetic resonance （NMR） , then react with OPAV which had been introduced thiol through N - Suceinimidyl - S - aeetylthioacetate （ SATA ）, human umbilical vein endothelial cells （HUVECs）were harvested in vitro and the cell were seeded in each group with density of 1 × 105 , CellTiter 96 AQueous One Solution Cell Proliferation Assay ,Hematoxylin and co- sin （HE） stain and scanning electron microscopy （SEM） were performed for each group to detect the HU- VECs growth 8 d after the seeding. Results 1HNMR （D20） ： 3.51 - 3.86 ppm belong to PEG peak, 4. 14 （4H, CH2OAr） , 5.80 - 6. 48 ppm belong to acrylate bond. SEM shows that DPAV modified with branched PEG become thickening, aftr HUVECs had grown for eight days, cell numbers in DPAV group was（0. 88 ±0. 03） × 105/cm2, while PEG - DPAV group was（ 1. 18 ±0. 04） ×105/ cm2. the cell numbers were higher in group PEG - DPAV as compared to those in group DPAV （ P 〈 0. 05 ）. HE and SEM obser- vations indicated that A confluent monolayer of endothelial cells was present on the valve surface in group PEG -DPAV at 8th d after the seeding, but not complete. Conclusion DPAV modified with branched PEG can improve the growth of endothelial cells, but cannot forming a complete fusion layer in the surface of decellularized valve scaffolds, so the PEGyalated DPAV can not achieve the desired endothelialization, and should be further modified.