| Prosthetic vascular and valve grafts seeded with endothelial cells have been used clinically for increasing patency and reducing grafts thrombogenicity. As one kind of cardiovascular implants, intravascular stent may induce thrombosis and in-stent restenosis at the same as decreasing restenosis ratios after percutanous transluminal angiography. Thrombosis is due to endothelium injury and stent as foreign body, in-stent restenosis is mainly due to neointima. It is possible that seeding endothelial cells on intravascular stent to prevent or decrease thrombosis and in-stent restenosis. So it is necessary to conduct the basis research of cells seeding for clinical apply. There are many problems which need to be solved on endothelial cells seeding stent. The purpose of this study is to strengthen the function of endothelial cells with gene modification, and study the change of endothelial cells in imitate circulation system in vitro. The results of the study will provide experiment support for endothelial cells seeding vascular stent and other cardiovascular implants. 1.Establishment of ECV-304 line transfected by VEGF gene with liposome, and influence of transfection of VEGF on endothelial cells function.Objective: To establish ECV-304 line expressing VEGF stably with VEGF gene transfecting, to assay the effect of VEGF transfection on the morphology, adhesion, proliferation and secretion of NO of endothelial cells .Methods: After extracting, purifying, identifying the plasmid PCD2-VEGF121 including VEGF gene, ECV-304 was transfected with the plasmid by lipofectAMINE, screened to monoclone formation with G418. Monoclone cells were detected whether they were transfected by RT-PCR and expressed the VEGF protein by immunohistochemistry. The secreted protein activity was assayed by Miles trial. The morphology and cytoskeleton fibrin and growth of transfected cells was compared to the normal cells. The adhesive ability of transfected cells was assayed with parallel-plate-flow-chamber. NO was assay with NO kit. Results: The results of RT-PCR and immunohistochemistry and Miles trial confirmed that ECV-304 expressing VEGF stably was established successfully, and the VEGF protein had biological activity. The morphology of transfectedcells elongated, and growth rate increased, cytoskeleton did not change. Secretion of NO was highest at 24 hours after subculture, but difference of NO secretion emerged only at 48 and 72 hours up to 96 hours. Conclusions:l.ECV-304 line expressing VEGF stably was established successfully.2. After transfected, the morphology of endothelial cells elongated which benefited endothelial cells stable adhesion ,and NO secretion increased, proliferation increased.2. The screen of substrates promoting endothelial cells adhesion and retentionObjective: To screen substrates for promoting endothelial cells adhesion and retention. Methods: The same number of endothelial cells was added to 96 hole culture plate which were precoated with poly-1-lysine and collagenIV and fibronectin in the concentration of 0. K 5 ^ 1CK 20 u g/ml respedtively. After 2 hours, cell numbers were counted under microscopy for four times. The characters of intravascular stents were detected in X ray fluorescence energy-chart instrument and transmission electron microscopy. The cover of substrates on the intravascular stents was observed with transmission electron microscopy after stents were treated with fibronectin and poly-1-lysine. Endothelial cells were cultured with stents in one rotation in ten minutes, and placed for one hour calmly after continued for 1 hour. Endothelial cells were counted after digested from the stents, the adhesive capability of two substrates were compared, the adhesion of endothelial cells on the stents were observed under transmission electron microscopy, and the growth of endothelial cells on the stents was detected. Endothelial cells were cultured on the plate which was precoated with poly-1-lysine and collagen IV and fibronectin in the optimum concentration respectively, after mixing to...
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