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

摘要

Objective To investigate the endothelialization performance of decellularized porcine aortic valve (DPAV) modified with branched polyethylene glycol.Methods Synthesis of 4-arm-Polyethyl glycol (PEG)-acrylate,characterized by 1H nuclear magnetic resonance (NMR),then react with DPAV which had been introduced thiol through N-Succinimidyl-S-acetylthioacetate (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(R) AQueous One Solution Cell Proliferation Assay、Hematoxylin and eosin (HE) stain and scanning electron microscopy (SEM) were performed for each group to detect the HUVECs growth 8 d after the seeding.Results 1 HNMR (D2O):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 observations 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.%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-acrylate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1×105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 1HNMR(D2O):3.51～3.86 ppm处PEG信号峰,4.14(4H,CH2OAr),5.80 ～6.48 ppm处丙烯酸酯双键.SEM可见枝状化PEG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/cm2,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,部分可见细胞间连接紧密形成一层近似融合的细胞层,但细胞紧密连接不连续.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,但却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.