| Object Multiple studies showed a remarkable reduction in the rate of restenosis and need for new revascularization procedures associated with DES compared to conventional bare metal (BM) stents. However, the long-term safety of these devices is far less certain. Recent reports pointed to the risk of late stent thrombosis and delayed arterial healing, which may be increased in DES using a polymer-based coating for retardation of drug release. There is evidence that application of polymers may lead to hypersensitivity reactions and, in few cases, late cardiac death. Furthermore, the issue of late-stent thrombosis in DES on the basis of delayed arterial healing, particularly after discontinuation of antiplatelet therapy, is currently subject to ongoing discussions. With the current technology, major hurdles to long-term success have not yet been taken. Thus, the development of novel stent designs with different anti-proliferative drugs, biodegradable polymers, or bioabsorbable stent matrices are being investigated. Cellular repressor of E1A-stimulated genes (CREG) is a recently identified secreted glycoprotein that antagonizes transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. CREG has a critical role in maintenance of smooth muscle cells in a mature, growth-quiescent state in the normal artery, and the downregulation of CREG contributes to neointimal hyperplasia after vascular injury. Our study also found that CREG facilitates migration and proliferation of ECs and contributes to the maintenance of vascular homeostasis. In this study we investigate the in vitro pharmacokinetic of nanoporous CREG eluting-stent.To evaluate the efficacy and safty of nanoporous CREG-eluting stent (CREGES) in inhibiting neointima proliferation in porcine coronary model.Methods 1: The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot. 2: For the absorption of the CREG protein by the nanoporous stent ,the stents were totally immersed and kept vertical in a 0.5 mg/mL ,1.0 mg/mL and 1.5 mg/ml solution of CREG protein in a phosphate buffer at 37℃in a 1.5 mL polypropylene (Eppendorf tube). These stent wires were further transferred in a 5% solution of BSA at 37℃for 1 h, followed by three rinsing cycles in PBS pH 7.4, 2 min each. Then, they were immersed in a 20μg/mL solution of fluorescein rabbit anti-huamn IgG in PBS, pH 7.4, at 378C for 30 min, followed by three rinsing cycles in PBS, pH 7.4, 2 min each. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from to investigate the cellspecific pharmacokinetic effect of CREG protein and rapamycin. 3: The nanoporous bare metal stent(BMS), nanoporous CREG eluting-stent(CREGES) and sirolimus-eluting stent(PARNTER) were implanted in left anterior descending coronary,left circumflex coronary and right coronary artery of fourty porcines in random. And after 7, 14 and 28-day,animals were sacrificed for histomorphologic and pathologic score analysis.Results: The lysates of 293F cells transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices, the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Stents eluting the CREG protein were tested for their adsorption characteristics by radioisotope technique with 125I labeled CREG protein. The amount of CREG protein adsorbed onto the nanoporous bare metal stent was dependent on the concentration and duration of immersion in the solution.We have tested three different concentrations for 48h.Maximal CREG protein binding was therefore defined as the amount of agent bound to stent wires after 48 hours immersion in a 1.5 mg/ml solution. We implant the CREG eluting stents and 316L stainless steel stents the control in pig model to study the bio-security validity of prevention and cure ISR by sliding microtome,SEM,transmission electron microscope(TEM),immunohistochemi- stry,tissue stain and biochemistry methods. The results suggests the CREG eluting stents can inhibit the thrombosis and neointimal hyperplasia by accelerating the endothelialization of the stent surface and inhibit the smooth muscle cell proliferation.Conclusion The nanoporous CREG-eluting stent in this study represents a novel promising device in preventing in-stent restenosis... |
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