肝素修饰改善去细胞化猪肝脏支架材料的生物相容性

发布时间：2018-06-14 09:04

本文选题：肝脏 + 去细胞　；参考：《泸州医学院》2014年硕士论文

【摘要】：目的：文章通过肝素修饰的方法，对改善作为组织工程支架材料的去细胞化猪肝脏的生物相容性进行了初步研究，，以期望能将此方法用于改善组织工程肝脏材料的生物相容性，以及为其他血液接触的组织工程材料改善生物相容性提供一个参考办法。方法：我们用不同的肝素修饰方法制作了不同种类的肝素化去细胞化猪肝脏支架材料。t-LBL （tissue immobilized with heparin by ionic binding vialayer-by-layer self-assembly technique）支架材料是通过去细胞化猪肝脏支架经肝素层层自组装离子结合的方式完成的。t-MPA(tissueimmobilized with heparin by covalent binding via multi-point attachment)支架材料是通过去细胞化猪肝脏支架经肝素多点连接共价结合的方式完成的。t-EPA(tissue immobilized with heparin by covalent bindingvia end-point attachment)支架材料是通过去细胞化猪肝脏支架经末端连接共价结合的方式完成的。文章通过甲苯胺蓝染色（Toluidine bluestaining）检测、甲苯胺蓝比色法（toluidine-blue colorimetric method）检测、H E染色（Hematoxylin Eosin staining）检测、凝血时间检测（coagulation time tests）、血小板粘附实验（Platelet adhesion）、血浆复钙时间检测（Plasma recalcification time）、材料上HepG2细胞培养实验（Cell culture of HepG2）、MTT分析、溶血度检测实验（Haemolysisratio measurement）和扫描电镜（scanning electron microscopy，SEM）检测评估了肝素化去细胞化猪肝脏支架材料的肝素化效果、抗凝血性能、细胞相容性。结果：t-LBL, t-MPA和t-EPA肝素化去细胞猪肝脏支架材料拥有不同的肝素含量。肝素化的去细胞化猪肝脏支架材料比未肝素化的去细胞猪肝脏支架材料具有更好的抗凝血性能。和t-LBL, t-MPA肝素化去细胞支架材料比较，t-EPA肝素化去细胞支架材料拥有更多肝素、肝素释放速度更慢而保留的肝素比例更多。和t-LBL, t-MPA肝素化去细胞猪肝脏支架材料比较，t-EPA肝素化去细胞猪肝脏支架材料的凝血酶时间（the thrombin time，TT），凝血酶原时间（prothrombin time，PT），活化部分凝血活酶时间（activatedpartial thromboplastin time，APTT）超出了设备测量范围。而且，t-EPA肝素化去细胞猪肝脏支架材料的血浆复钙时间是肝素修饰各组支架材料中最长的。t-EPA肝素化去细胞猪肝脏支架材料比对照、t-LBL和t-MPA更能促进HepG2细胞在材料上增殖、分布、聚集和伸展。结论：通过末端（end-point attachment，EPA）连接肝素共价结合修饰的去细胞化猪肝脏支架材料较层层自组装（layer-by-layerself-assembly technique，LBL）和多点结合（multi-point attachment，MPA）肝素化去细胞化猪肝脏支架材料获得了良好的抗凝性和细胞相容性。鉴于在温和的水溶液条件下完成肝素修饰的去细胞化猪肝脏支架材料制备和其在肝脏组织工程中拥有良好的抗凝性能，本研究为制备其他组织工程用的具有良好抗凝性能的与血液接触的支架材料提供了一种参考方法。
[Abstract]:Objective: to improve the biocompatibility of decellated porcine liver as tissue engineering scaffold material by heparin modification method in order to improve the biocompatibility of tissue engineering liver materials. And to provide a reference for other blood contact tissue engineering materials to improve biocompatibility. Methods: different kinds of heparinized porcine liver scaffolds. T-LBL tissue immobilized with heparin by ionic binding vialayer-by-layer self-assembly techniques were prepared by different heparin modification methods. The. T-MPAtissue with heparin by covalent binding via multi-point attachment material, completed by the method of assembling ion binding, is made by means of multipoint connection of heparin and covalent binding of porcine liver stents. T-EPA-tissue immobilized with heparin by covalent bindingvia end-point attachment materials are fabricated by means of the. T-EPA-tissue immobilized with heparin by covalent bindingvia end-point attachment material, which is made by means of decellulization of porcine liver stents by means of multipoint connection and covalent binding of heparin. The acellular porcine liver scaffold was covalently linked to the end of the liver. Toluidine bluestainingwas detected by toluidine blue staining, and Hematoxylin eosin stainingwas detected by toluidine blue colorimetry and toluidine blue colorimetric method. Coagulation time test, platelet adhesion test, plasma recalcification assay, cell culture of HepG2 cell culture assay were used. The hemolysis ratio measurement and scanning electron microscopy (SEM) were used to evaluate the heparin effect, anticoagulant performance and cytocompatibility of heparinized porcine liver scaffolds. Results different heparin contents were found in 10% t-LBL, t-MPA and t-EPA heparinized porcine liver scaffolds. Heparinized porcine liver scaffold has better anticoagulant performance than non-heparinized porcine liver scaffold. Compared with t-LBLand t-MPA heparinized scaffolds, the heparinized stents had more heparin, slower heparin release rate and more heparin retention. Compared with t-LBLand t-MPA heparinized porcine liver scaffolds, the thrombin time and prothrombin time of t-EPA heparinized porcine liver scaffold materials were higher than those of the thrombin thromboplastin time, and the activated partial thromboplastin time (APTT) exceeded the measuring range of the equipment. Moreover, the plasma recalcification time of heparinized porcine stents was the longest. T-EPA heparinized porcine liver scaffolds could promote the proliferation and distribution of HepG2 cells. Gather and stretch. Conclusion: the anticoagulant and cytocompatibility of heparin covalently modified porcine liver scaffolds by end-point attachment (EPA) is better than that of layer-by-layerself-assembly technique (LBLL) and multi-point attachment MPA-heparinized porcine liver scaffolds. In view of the preparation and anticoagulant properties of heparin modified acellular porcine liver scaffolds in mild aqueous solution, This study provides a reference method for the preparation of other scaffolds with good anticoagulant properties for tissue engineering.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08;R657.3

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