内皮祖细胞捕获表面构建及其功能评价

发布时间：2018-03-29 23:05

本文选题：血液相容性　切入点：内皮化　出处：《西南交通大学》2012年博士论文

【摘要】：内皮祖细胞(Endothelial progenitor cell, EPC)对于血管支架植入后损伤血管内皮细胞层的重建具有重要作用。目前,新开发的内皮祖细胞捕获血管支架相对于药物洗脱支架和裸支架具有更好的治疗效果,但仍存在一些问题：1)由于未考虑抗凝血修饰,血栓仍可能导致治疗失败；2)血液中内皮祖细胞数量稀少,且在血流剪切力作用下,难以实现在支架植入部位的快速粘附,阻碍了内皮细胞层快速重建；3)目前血管支架材质为不可降解金属,其长期存在于血管中扰乱了血管舒缩等正常生理功能,为晚期血栓和晚期支架内再狭窄埋下隐患。 EPC对心肌梗死患者的坏死心肌细胞再生和心肌功能恢复具有重要作用,但由于其在病变部位粘附困难,难以实现对病变部位的有效治疗,所以将EPC定向运输到指定部位的方法学研究具有重要的学术意义和潜在的应用价值。针对以上问题,本文采用：1)在硅烷修饰的纯钛表面接枝双羧基聚乙二醇后,再进一步共价固定CD34抗体,构建出具备优异血液相容性和特异性捕获EPC的多功能生物活性表面；2)制备了具有可以特异性结合EPC的MNP,用于将结合MNP的EPC,在外加磁场辅助下,富集于磁靶向部位(支架植入处或是心肌梗死区域)并加速EPC粘附,实现病变部位快速EPC覆盖；3)研究了可生物降解的铁支架在MNP和外磁场辅助下表面快速粘附EPC的可能性。具体内容如下： 1.纯钛依次经碱热处理和硅烷修饰,在表面获得大量的高反应活性的氨基,并通过氨基-羧基缩合将双羧基的聚乙二醇接枝在纯钛表面,通过反应条件的控制,获得不同链长和不同链密度PEG接枝表面,通过多种检测手段证明表面构建成功；详细研究了PEG链长和链密度与表面生物相容性的关系,结果显示具有高链密度(3chains/nm2)和长链(PEG4000)的PEG接枝表面可以更有效抑制纤维蛋白原、血小板、平滑肌细胞和巨噬细胞的粘附。 2.在对PEG接枝表面优选后,通过PEG上的羧基与CD34抗体上的氨基缩合,实现CD34抗体在表面的固定,多种检测手段证明表面构建成功,对构建表面的生物相容性的研究结果显示,在PEG长链接枝的钛表面连接抗体后,不但保持了优异的血液相容性和捕获EPC的能力,而且表现出对平滑肌细胞粘附的抑制作用和更低的炎症反应。 3.通过水热共沉淀法合成出表面包裹双羧基聚乙二醇的Fe304,并进一步共价连接CD34抗体,制备出具有良好磁响应性和对EPC特异性识别的磁性纳米粒子(MNP),结果显示该MNP以100μg/mL的使用浓度为宜,该浓度下EPC在磁场施加部位粘附数量成倍提升而平滑肌细胞粘附被抑制,且未引发凝血和溶血,动物体内实验证明其在体内亦具有对EPC的特异性结合能力和对磁场的响应能力,并不会被免疫系统快速清除。 4.可降解铁支架在体内出现了严重的再狭窄,但其降解特性严重阻碍了表面生物化修饰,故难以解决再狭窄问题,通过MNP和外加磁场辅助,保证其降解行为不受影响的前提下有效实现了EPC在表面的快速粘附,结果显示高磁场强度,长磁场作用时间,高EPC浓度可以增加EPC在铁表面粘附量。对于铁支架的应用具有重要参考价值。
[Abstract]:Endothelial progenitor cells ( EPC ) play an important role in the reconstruction of vascular endothelial cells after vascular stent implantation . Currently , newly developed endothelial progenitor cells capture vascular stents with better therapeutic effects relative to drug eluting stents and bare stents , but there are still some problems : 1 ) Thrombus may still lead to treatment failure due to the absence of anticoagulant modification ;
2 ) the number of endothelial progenitor cells in the blood is sparse , and under the action of blood flow shearing force , it is difficult to realize the rapid adhesion of endothelial progenitor cells at the implantation site of the stent , thereby inhibiting the rapid reconstruction of the endothelial cell layer ;
3 ) At present , the vascular stent material is non - degradable metal , and the long - term presence of the vascular stent material is disturbed by the normal physiological function of the vasomotor and the like in the blood vessel , and the hidden trouble is hidden in the late thrombosis and the late stent restenosis .

EPC plays an important role in the regeneration of necrotic myocardial cells and the recovery of myocardial function in patients with myocardial infarction , but it is difficult to achieve effective treatment of the lesion site due to the difficulty of adhering to the lesion site , so the methodological research for transporting the EPC to the designated site has important academic significance and potential application value .

In view of the above problems , 1 ) After grafting the double carboxyl polyethylene glycol on the silane - modified pure titanium surface , we further covalently immobilize the CD34 antibody to construct a multifunctional bioactive surface with excellent blood compatibility and specific capture EPC .
2 ) preparing MNP which can specifically bind to the EPC , and is used for enriching the EPC of the MNP , and enriching the EPC with the magnetic targeting site ( the stent implantation or the myocardial infarction area ) under the assistance of the external magnetic field and accelerating the EPC adhesion to realize the rapid EPC coverage of the lesion site ;
3 ) The possibility of rapid adhesion of the biodegradable iron stent to the surface of MNP and external magnetic field is studied . The specific contents are as follows :

1 . the pure titanium is sequentially subjected to alkali heat treatment and silane modification , a large amount of high - reactive amino groups are obtained on the surface , and the polyethylene glycol of the bicarboxyl group is grafted on the surface of the pure titanium by amino - carboxyl condensation , the PEG grafting surfaces with different chain lengths and different chain density are obtained through the control of the reaction conditions , and the surface construction success is proved by various detection means ;
The relationship between PEG chain length and chain density and surface biocompatibility was studied in detail . The results showed that PEG grafted surface with high chain density ( 3chains / nm2 ) and long chain ( PEG4000 ) could inhibit the adhesion of fibrinogen , platelets , smooth muscle cells and macrophages more effectively .

2 . After the PEG grafting surface is preferred , the immobilization of the CD34 antibody on the surface is realized by the amino condensation on the carboxyl group on the PEG and the amino group on the CD34 antibody , and various detection means prove that the surface construction is successful , and the research result of the biocompatibility of the construction surface shows that the excellent blood compatibility and the ability of capturing the EPC are maintained after the antibody is attached to the titanium surface of the PEG long linking branch , and the inhibition effect on the adhesion of the smooth muscle cell and the lower inflammatory reaction are shown .

3 . The magnetic nanoparticles ( MNP ) with good magnetic responsiveness and EPC - specific recognition were synthesized by hydrothermal coprecipitation method . The results showed that the concentration of EPC was better than that of 100 渭g / mL . The results showed that the adhesion of EPC to 100 渭g / mL was higher than that of EPC under the concentration of 100 渭g / mL .

4 . The degradable iron stent has serious restenosis in the body , but its degradation characteristics seriously hinder the surface biological modification . Therefore , it is difficult to solve the problem of restenosis . By means of MNP and external magnetic field , the rapid adhesion of the EPC to the surface is effectively realized . The results show that the high magnetic field strength , the long magnetic field action time and the high EPC concentration can increase the adhesion amount of the EPC to the iron surface .

【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.0

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