【作者】 陶运明；

【导师】 胡铁辉；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章二羟基铁/肝素复合物纳米修饰去细胞血管基质的制备和生物相容性评价目的：去细胞异种血管植入体内存在血栓形成的风险。本研究旨在采用二羟基铁/肝素复合物(DHCs)修饰去细胞血管基质以提高其生物相容性。评估其作为一种新型肝素修饰去细胞异种血管方法的可行性。方法：使用二羟基铁作为交联剂,采用层层自组装技术将二羟基铁(DHI)和肝素交替固定在BJV表面,构建一种新型的DHCs抗凝表面。通过甲苯胺蓝比色法检测剩余肝素浓度而获得每次自组装结合肝素的量；扫描电镜(SEM)和组织切片甲苯胺蓝染色检测肝素和二羟基铁层层自组装修饰BJV (LBL-BJV)的表面微结构；拉力试验检测其生物力学性能；洗脱试验检测DHCs与BJV结合的稳定性；抗凝血活性检测、血小板粘附实验、内皮细胞增殖实验和抗钙化实验检测LBL-BJV的生物相容性。结果：甲苯胺蓝比色法显示每组装一次约有808±86μg/cm2肝素固定在BJV表面；扫描电镜显示DHCs是均匀的包裹在胶原纤维表面并形成纳米膜；甲苯胺蓝组织切片染色提示肝素主要结合在BJV的表面；拉力试验提示实验组的生物力学稳定性显著地提高；洗脱试验提示DHCs稳定地结合在BJV的表面并持续缓慢的释放肝素；抗凝血活性检测显示实验组PT和APTT明显高于正常值范围；实验组材料表面的血小板数明显低于对照组,每10000μm2LBL-BJV和DC-BJV表面血小板计数分别为8±4和48±16；内皮细胞增殖实验提示经过7天的培养,实验组和对照组材料表面内皮细胞数相似；在皮下包埋4周时,LBL-BJV和DC-BJV中钙离子的含量分别为8.5±1.9μg/mg和26.6±3.7μ/mg;8周时,LBL-BJV和DC-BJV中钙离子的含量分别为21.5±6.8μg/mg和112.6±16.9μg/mg。结论：DHCs牢固地结合在DC-BJV表面,形成抗凝表面,并长时间的缓慢释放肝素。DHCs纳米修饰能够提高去细胞血管基质的生物力学稳定性和生物相容性。第二章二羟基铁/低分子肝素纳米颗粒的制备与性能评价目的：低分子肝素钠(LMWH)的半衰期短,维持抗血栓活性时间短。本研究旨在制备一种具有抗凝血活性的、能缓释LMWH的DHI/LMWH纳米颗粒(DLN),并评估其作为一种新型抗凝血药物的可能性。方法：在超声振荡或磁力搅拌条件下,将DHI溶液加入LMWH溶液而形成DLN溶液；并将两种方法在不同的DHI/LMWH质量比条件下,检测两种条件下形成的纳米颗粒形状、粒径、zeta电位和包封率；红外吸收光谱检测DLN与LMWH两者的红外吸收光谱的差异。结果：在超声振荡条件下形成的纳米颗粒平均粒径小于100nm,而在磁力搅拌条件下形成的纳米颗粒平均粒径大于100nm；两种处理方法对纳米颗粒的zeta电位和包封率都没有影响：zeta电位均为负值；包封率与DHI/LMWH的质量比呈正相关；当DHI/LMWH质量比低于1.2时,形成纳米颗粒的平均粒径低于100nm,当DHI/LMWH的质量比大于1.2时,形成的纳米颗粒的粒径超过100nm,并出现微米级颗粒;SEM图片显示DLN呈矩形晶体状结构；DLN与LMWH的红外吸收光谱大体相似,但DLN的红外吸收光谱中出现1732cm-、1549cm-、690cm-、423cm-四处吸收峰；DLN在PBS中稳定存在并缓慢释放：第一天释放量稍大,约占总量的5.1%,其后是均匀释放,经4周的震荡,LMWH的释放量约占总量的43.8%。结论:DHI和LMWH能够在超声振荡条件下形成具有缓释功能的抗凝纳米颗粒,DLN有望成为一种新型的纳米抗凝药物。第三章二羟基铁/低分子肝素纳米颗粒-VEGF修饰促进去细胞血管基质的生物相容性和内皮化的研究目的：异种移植血管存在生物相容性差和血管内皮化不足等缺陷,为了促进去细胞血管基质的生物相容性和内皮化,采用DLN-VEGF修饰去细胞血管基质表面,评估其作为一种修饰去细胞异种血管方法的可行性。方法：先使用DHI和LMWH预处理去细胞牛颈静脉(DC-BJV),通过DHI和LMWH之间作用力将DLN固定到去细胞血管基质的表面,再通过LMWH将VEGF固定到去细胞血管基质的表面,构建一种二羟基铁／低分子肝素纳米颗粒-VEGF修饰的去细胞牛颈静脉(DLN-VEGF-BJV);扫描电镜(SEM)和组织切片甲苯胺蓝染色检测DLN-VEGF-BJV的表面微结构；拉力试验检测其生物力学性能；洗脱试验检测DHI与BJV结合的稳定性；血小板粘附实验、内皮细胞增殖实验和抗钙化实验检测其的生物相容性；体外内皮细胞增殖实验检测其促内皮细胞生长、增值能力。结果：扫描电镜显示DLN广泛分布DLN-VEGF-BJV血管基质表面的纤维表面和纤维缝隙间；甲苯胺蓝组织切片染色提示DLN主要结合在BJV材料的外表面；拉力试验提示实验组的生物力学稳定性有显著性提高；免疫组化提示VEGF结合在血管基质的表面；释放试验提示LMWH和VEGF能够缓慢的从其表面释放；并具有抗抗血小板粘附,每10000μm2DLN-VEGF-BJV和DC-BJV表面的血小板计数分别为12±4和48±16；内皮细胞增殖实验实验提示经过3天和7天的培养,DLN-VEGF-BJV表面内皮细胞数明显大于DC-BJV表面内皮细胞数；皮下包埋4周时,DLN-VEGF-BJV和DC-BJV钙离子的含量分别为8.1±1.7和26.6±3.78周时,DLN-VEGF-BJV和DC-BJV钙离子的含量分别为23.5±6.1μg/mg和112.6±16.9μg/mg结论：DLN能够被稳定地修饰在去细胞血管基质的表面,并能够提高去细胞血管基质的生物力学稳定性、抗凝、抗血小板粘附、抗钙化和促进内皮细胞的生长和增殖,有望成为一种新型的去细胞异种移植血管的修饰方法。更多还原

【Abstract】 Part I Preparation of dihydroxy-iron/heparin complexes nanomodified decellular vascular matrix and evaluation of its biocompatibilitiesObjective:There are risks of thrombosis after decellular xenograft implantation, the purpose of this study was to improve the biocompatibilities of decellular vascular matrix by dihydroxy-iron/heparin complexes (DHCs) nanomodification and evaluate the possibility of method which as a novel heparin modification for decellular xenograft.Methods:A novel thrombo-resistant surface for decellular xenograft had been developed by alternating linkage of dihydroxy-iron and heparin to decellular bovine jugular vein (DC-BJV) using dihydroxy-iron (DHI) as crosslinker. Toluidine blue colorimetric method was used to measure residual content of heparin solution after each assembly cycle, and the amount of linked heparin was calculated. Surface characterization of dihydroxy-iron/heparin layer-by-layer-assembly modified BJV (LBL-BJV) was assayed using scanning electron microscopy (SEM) and toluidine blue staining. Its biomechanical stability was detected by tensile test. The binding force of DHCs on surface of BJV was evaluated by shaken-wash test. And its biocompatibilities were detected using antithrombogenicity, platelet adhesion and cytotoxicity assay.Results:Toluidine blue colorimetric method showed the amount of linked heparin was about808±86μg/cm-1per assembly-cycle. SEM images proved DHCs were uniformly linked to and formed nanoscale films around the fibrils of DC-BJV. Toluidine blue staining histology pictures showed DHCs were mainly linked to DC-BJV surface. Washing test proved DHCs were firmly linked to BJV and sustainedly released heparin for a long time. Tensile test showed that biomechanical stability was increased. Hemocompatibility evaluations showed that PT and APTT of all trial groups were above the normal reference ranges, and mean platelet count per10000μm2area was8±4for LBL-BJV vs.48±16for DC-BJV. The endothelial cells (ECs) proliferation test showed the number of ECs on luminal surface of LBL-BJV was very similar to DC-BJV at7-day incubation. Calcium content assay showed the mean calcium content was8.5±1.9μg/mg dry weight for LBL-BJV vs.26.6±3.7μg/mg dry weight for DC-BJV at30days and21.5±6.8μg/mg dry weight for LBL-BJV vs.112.6±16.9μg/mg dry weight for DC-BJVs at60days, respectively.Conclusions:DHCs nanomodification improves biomechanical stability and biocompatibilities of decellular xenograft. Part Ⅱ Preparation and in vitro evaluation of dihydroxy-iron/low-molecular-weight-heparin nanoparticleObjective:The half-life of low molecular weight heparin (LMWH) is short, and its antithromboticity doesn’t maintain for a long time. The purpose of current study was to prepare thrombo-resistant DHI/LMWH nanoparticles (DLN) which sustainedly released LMWH and evaluate the possibility of which as a novel anticoagulatant drug.Methods:A novel thrombo-resistant DLN was prepared by LMWH and DHI under ultrasonic oscillation or magnetic stirring. The shape, size, zeta potential and encapsulation efficiency of DLNs were detected, and the difference of DLN and LMWH were analyzed by infrared absorption spectrum.Results:Average size of DLN was less than100nm under ultrasonic oscillation, and average size of dihydroxy-iron/low-molecular-weight particle was more than100nm under magenic stirring. But ultrasonic osscillation and magenic stirring did not affect the zeta potential and encapsulation efficiency of nanoparticles, their values of zeta potential were negative value, and their encapsulation efficiency was positively correlated to weight-ratio of DHI/LMWH. The average size of DLN was less than100nm when the weight-ratio of DHI/LMWH was less than1.2, and their average size was more than100nm and formed micro-particles when the weight-ratio of DHI/LMWH was more than1.2. SEM immages showed the shape of DLN was rectangle. Compared infrared absorption spectrums of DLN and LMWH, there were1732,1549,690and423cm-absorbtion peak appeared in infrared absorption spectrum of DLNs. DLNs were sustainedly released LMWH in PBS for a long time.Conclusions:DLN can be prepared via LMWH and DHI under ultrasonic oscillation, and it is a novel thrombo-resistant nanoparticle. Part Ⅲ The study of dihydroxy-iron/heparin-VEGF nanoparticles modification improves biocompatibilities and endothelialization of decellular vascular matrixObjective:Poor biocompatibility and lack of endothelialization are limitations of decellular xenograft, the purpose of current study was to improve the biocompatibilities and endothelialization of decellular vascular matrix via DHI/LMWH-VEGF nanoparticle modification, and evaluate the possibility of method which as a novel modification for decellular xenograft.Methods:A DHI/LMWH-VEGF nanoparticle modified BJV (DLN-VEGF-BJV) was developed as follows:DC-BJV was preteated by DHI and LMWH, then DHI/LMWH nanoparticles (DLNs) were linked to sueface of DC-BJV via DHI and LMWH, and then VEGF was immobilized to surface of DLN via LMWH. Its surface characterization was assayed using SEM and toluidine blue staining; its biomechanical stability was detected by tensile test; the binding force of DLNs on surface of BJV was evaluated by shaken-wash test; its biocompatibilities were detected using platelet adhesion, cytotoxicity and anti-calcification assay; and its stumilating-endothelial-cell-growth was evaluated by endothelial cells proliferation assay.Results:SEM images proved DLNs were uniformly linked to the fibrils of BJV. Toluidine blue staining histology pictures showed DLN were mainly linked to BJV surface. Shaken-washing test proved DLN-VEGF-BJV sustainedly released LMWH and VEGF for a long time. Tensile test showed that biomechanical stability was increased. Static platelet adhesion assay results showed that the modified BJV drastically decreased platelet adhesion, and the mean platelet count per10000urn2area was12±4for DLN-VEGF-BJV vs.48±for DC-BJV. The Endothelial cells proliferation in vitro assay showed ECs could adhere and proliferate on luminal surface of both DLN-VEGF-BJV and DC-BJV, and the number of ECs on the luminal surface of DLN-VEGF-BJV was more than that on DC-BJV at3-day and7-day incubation, respectively. Calcium content assay showed the mean calcium content was8.1±1.7μg/mg dry weight for DLN-VEGF-BJV vs.26.6±3.7μg/mg dry weight for DC-BJV at30days and23.5±6.1μg/mg dry weight for DLN-VEGF-BJV vs.112.6±16.9μg/mg dry weight for DC-BJVs at60days, respectively. Conclusions:The DHI/LMWH-VEGF nanoparticles modification improves biomechanical stability, antithrombogenicity, anticalcification efficacy and endothelialization of decellular vascular matrix.更多还原