| 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.
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