【作者】 唐朝君；

【导师】 王贵学；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【摘要】 冠状动脉内植入支架已成为治疗冠心病的重要手段。然而,支架植入不能抑制新生内膜增生,支架内再狭窄(In-stent restenosis, ISR)的发生率仍有(13-20)%。目前,防治支架内再狭窄的研究热点在于药物洗脱性支架,而该支架植入后出现的一些问题已引起广泛关注:如晚期支架血栓形成,支架涂层材料的不良病理作用,内皮化延迟、动脉瘤、支架贴壁不全等。促进损伤血管愈合、加速支架完全再内皮化过程是解决问题的一条有效途径。因此,内皮细胞(Endothelial Cell, EC)在再狭窄形成过程中的作用越来越受到重视。内皮细胞移植能替代、修复受损内皮的功能并抑制内膜过度增生。体外培养的内皮细胞由于生长环境的改变,导致生长缓慢和其他生物特性的丢失,结合基因治疗,有望通过对体外培养的内皮细胞稳定转染生长因子,提升细胞的增殖能力;结合动态培养,有望促进内皮细胞在血管内支架表面的粘附和生长;结合模拟体内循环系统实验,有望控制细胞支架在输送过程中的损耗,保障动物实验结果的真实可靠性。本研究就是基于上述思路开展的应用基础研究,即转基因内皮细胞支架的初期研究。在本课题研究研究中,采用阳离子脂质体DOTAP对传代培养的人脐静脉内皮细胞稳定转染血管内皮生长因子VEGF121;并以此转基因内皮细胞为研究对象,设计并制作了用于血管内支架表面种植细胞的旋转培养装置,通过对旋转速度、旋转时间、细胞种植密度和重复旋转的次数这些影响因素的分析,寻找出了粘附效果最好的条件组合;用超声雾化喷涂法方法制备用于细胞粘附的蛋白涂层,并用扫描电镜和能谱测试仪检测制备好的蛋白涂层;根据得到的最佳旋转培养条件制备转基因内皮细胞包被支架,通过扫描电镜检测,旋转培养后血管内支架表面粘附的细胞数量和铺展情况,免疫荧光检测支架表面粘附的细胞表达VEGF蛋白的情况;体外流动腔实验检测经过传输的损耗和流动剪切力的作用后,转基因内皮细胞在血管内支架表面的粘附和生长情况;最后通过兔腹主动脉支架植入术的动物模型检测转基因内皮细胞包被支架的在体抗再狭窄和抗血栓的效果。研究结果如下:1)通过RT-PCR和免疫细胞化学鉴定,成功建立转VEGF基因人脐静脉内皮细胞系;2)通过对旋转速度、旋转时间、细胞种植密度和重复旋转的次数这些影响因素的分析,寻找出了粘附效果最好的条件组合,当细胞种植密度为1×105cells/ml,旋转时间为6h,旋转速度为0.4rpm时,旋转培养粘附的细胞数量最多;3)制备明胶浓度为2-6mg/ml,多聚赖氨酸为10μg/ml的混合溶液,用超声雾化喷涂法方法得到的蛋白涂层经检测在支架表面涂覆紧密、均匀;4)通过优化的旋转培养条件制备出转基因内皮细胞覆盖的血管内支架,在光学显微镜和扫描电镜下观察,细胞在支架表面粘附生长,荧光显微镜检测细胞高表达VEGF蛋白;5)体外流动腔实验检测发现,转基因内皮细胞在模拟体内传输过程中,将有部分细胞损失,但是在不同的切应力作用下,转基因内皮细胞又很快的恢复生长;6)动物实验结果发现与两个对照组相比,细胞包被支架均极显著的抑制了内膜增生和降低了支架内再狭窄。综上所述,本研究初步解决了血管内支架的细胞种植方法,提出一种组织工程血管内支架的实验室制备方法和检测方法,得到的相关实验结果为细胞种植防治血管内支架再狭窄的临床应用提供实验依据。内皮细胞种植支架预防再狭窄的研究已显示出诱人的前景,随着进一步的深入研究,相信将会开辟出一片新的领域。更多还原

【Abstract】 Vascular stent implant in coronary artery is critical treatment to the coronary heart disease. However, neointima hyperplasia still couldn’t be inhibited through stent embedding. The incidence rate of In-stent restenosis (ISR) is still to remain 13-20%. Most studies lay particular emphasis on drug-eluting stent (EDS), although the uncovered struts may be prone to late thrombosis after the discontinuation of antiplatelet therapy, and also the other potential complications such as delayed endothelialization, aneurysm because of the coating materials. So it may be an effective process to work out a solution by promoting re-endothelialization and injure healing of endothelial layer.Therefore, the integrity and functional activity of the endothelial monolayer play a crucial role in the prevention of thrombosis and in-stent restenosis. More and more researches illustrated that the coating endothelial cells (ECs) of stents would repair the endothelial layer of the injured arterial wall and suppress intima hyperplasy. ECs cultured in vitro may lost some biological characteristics and lead to slow growth due to the changes of environment. Combination of gene therapy is expected to enhance cell proliferation by stable transfection with vascular endothelial growth factor.Combination of dynamic culture is expected to promote the adherence and growth of endothelial cells on the vascular stent surface. Combination of the simulation experiments of circulatory system is expected to control the cells’loss in the process of stent implantation and improve the reliability of animal experiments results. This study is based on the above ideas in the application of basic research, the initial study of genetically modified endothelial cells coating stent.In this research, liposome DOTAP was used in the stable transfection experiment for vascular endothelial growth factor VEGF121 gene transfer to human umbilical vein endothelial cells (HUVECs) according to standard methods. Then the tansgenc ECs were used in the following experiments. A rotational culture device was designed for the cells seeding onto metallic stents surface through the analysis of these factors such as the rotation speed of rotation, planting density and repetition of the number of rotating, finding the best conditions for adhesion. Ultrasonic atomization spraying method was selected to prepare the protein coating, and scanning electron microscopy and energy spectrum were used for detection of protein coating. Transgenic endothelial cells was seeded onto the stents surface through the the best adhesion conditions we got. The morphology and growth of cells on the surface were observed respectively by SEM and the expression of VEGF was detected by fluorescence microscope. An extracorporeal circulation system was used to simulate the transfer process and check the cells adhesion and growth on the modified surface after stenting in vitro. Bare metal stent (BMS), protein coated stent and transgenic endothelial cells coated stent (ECS) were deployed in the infra-renal abdominal aorta to detect the efficiency on promoting re-endothelization and inhibiting in-stent restenosis in vivo.HUVECs line monoclonal cells with the stable expression of VEGF121 gene was establish through the detection of human VEGF121 protein expression by immunohistochemistry, and human VEGF121 RNA by RT-PCR. Cells grew on the surface of stents indeed through rotational culture, results showed that dynamic seeding was adequate when the rotational speed was 0.4 rpm, rotational time was 6h, cell density was 1×105 cells/ml. Hybrid solution was prepared with gelatin concentration of 2-6 mg/ml and poly-L-lysine concentration of 10 ng/ml. The protein coating was close and uniform through ultrasonic atomization spraying detected by SEM. Stent seeded with transgenic ECs were observed by SEM and fluorescence microscope. In vitro studies revealed that cells adhered on the surface of stents confertimly. And the covered rate of the surface achieved more than 90 percentages after 24 h. Immunofluorescence of VEGF121 also showed the high rate of coverage. Cells would lose a few after stent implantation in the flow system. Howere, cells kept increasing after 12 h. Transgenic ECs-coated stent was associated with a significant reduction in neointimal area and percentage stenosis, a significant promotion in re-endothelization on the surface of stent compared with bare metal stents and protein coated stent.To sum up, this study were initially solved the methods of cell seeding onto vascular stent surface, introduced a laboratory preparation and detection techniques for a vascular stent in tissue engineering. The results of experiments were provided evidences in the prevention and treatment of ISR in clinical application. Prevention of restenosis through endothelial cells planting has shown attractive prospects, with further in-depth study that will be opened up to a new area.更多还原