Development Of A20 Gene Modified Stents And Prevention To The Porcine Carotid Restenosis

BackgroundCerebrovascular disease is a common and frequently occurring illness with high case fatality rate and mutilation rate. In different types of cerebrovascular diseases, ischemic cerebrovascular disease is the most common. Minimally invasive interventional therapy within lumen of blood vessel has been applied in secondary prevention widely in recent years, decreasing incidence rate remarkably. It has the same curative effect as carotid endarterectomy(CEA) but less complication and unique advantages on intracalvarium arteries compared with CEA. However, in-stent restenosis influences its prostecdtive efficacy, especially in the process of small diameter cerebrovascular stent-assistant angioplasty. The damage of vascular endothelial cell triggered these series of pathology conditions; also, inflammation plays an important role in the process.With the high reproductive activity, endothelial progenitor cells can develop into endothelial cell under the vivo regional microenvironment, growing properly and performing the function as endothelial cell. A20 gene can promote EPCs to against causative agent and inhibit pathologic generation of vascular smooth muscle cells (SMC), besides; it can restrict the inflammation by decreasing mediators of inflammation. A20 gene can effectively help EPCs to resist pathogenic factors, restrain pathological proliferation of SMCs, and cut down partial inflammatory factors to restrain inflammatory reactions consequently. It is indicated in the prophase research that A20 can prevent histio-engineering blood vessel restenosis. In a word, stents for EPCs transfected by A20 gene is of great value in prevention of restenosis.Thus, we developed EPCs seeded stent at first, but because of the technology issue on cells seeded stent's delivery, we verified the EPCs transfected by A20 gene by animal experiment. This study aims at mechanism and elements of forming vascular restenosis and finding new vascular stent transfected by genes to prevent restenosis.Methods1. EPCs culture, identificationObtaining mononuclear cells from human umbilical cord blood by density gradient centrifugation, and induced cultured in M199 medium with adding VEGF, bFGF and other growth factor. Inverting microscope to observe cell growth and morphological changes; combining DiI-LDL uptake experiments and UEA-I to observe whether EPCs has the functional characteristics of endothelial cells or not; though CD34, CD133, KDR, vWF and the like to identify whether EPCs has the phenotype of endothelial cells; through TEM observation to find whether EPCs has endothelial cell ultrastructure;2. Development of EPCs seeded stentIdentification of EPCs transfected by A20 gene; processing of CD34 antibody coupled with stent (B group), A group was the control group; observed cell changes under CLSM and AMRAY (American) electron scanning microscope after EPCs seeded stent through the flow of chamber.3. Experiment research on restenosis prevention and treatment of endovascula stent transfected by A20 geneA20 gene-modified stent processing; in vivo implant into carotid artery of pigs, the control group pCDNA3.1 void vectors as A group, A20-modified stent as B group; test of A20 expression; adopting of the following indicators for judging the effectiveness of the two groups: Carotid angiography; histopathological analysis and pathological integration; CD31 and PCNA immunohistochemistry tests; apoptosis detection; electron microscopy of endothelial cells and smooth muscle cells.Results1. Identification of EPCs culture1.1 After cells in vitro culture 2 - 3 d, there are a small number of cell pustute, with varied hapes like short spindle, polygonal shape, etc.; after 7 - 9 d, cobblestone-like cells colonies appeared, with forming the clear cloning; continue to culture 3 - 4d, colonies gradually dispersed; when culture fluid re-suspended, continue to culture cells 4-5d until they gradually fused, showing a typical "cobblestone-like"appearance.1.2 EPCs ultrastructure: transmission electron microscopy observation showed the characteristics WP within EPCs, it can be seen at the same time a large number of phagocytic vesicles, mitochondria, endoplasmic reticulum and other cell organelles.1.3 Culture EPCs by immunochemical staining: In early phase, expression of CD34, CD133, KDR were positive; three weeks later, expression of CD34, KDR, vWF were positive, but expression of CD133 was negative.1.4 Detection of biological function: Double staining positive cells, uptaking DiI-acLDL and combining FITC-UEA-I, were seen as being differentiated EPCs.2. Development of EPCs seeded stent2.1 A20 transfected EPCs successfully, and it is positive by immunohistochemistry and Western-blot testing.2.2 The growth situation of cells above the tentsObserved under CLSM and AMRAY (American) electron scanning microscope after 7-day culture: 1.) both A and B groups of stents can be completely covered by cells, wherein in-stent endothelialization, photolysis of LDL show that favorable biological functions are all achieved. 2.) However, there is denudation of cells on the stents for both Group A and Group B after being rinsed in a flow chamber. Cells of Group A diminish remarkably in a scattered distribution and fail to cover the surface of a stent. On the contrary, cells of Group B remains covering the most part of a stent despite denudation. From these two results, while keeping the total number of cells of Group B is much exceeding that of Group A(P<0.01).3. Experiment research on restenosis prevention and treatment of endovascula stent transfected by A20 gene3.1 Detection of A20 expression: 3 days after implantation of vascular stents,A20 expressed in the regional area of blood vessel.3.2 Angiography: 3 months stenting, stent restenosis in the control group and experimental group had statistically significant differences (37.93%±2.11%, 15.03%±1.95%, P <0.01).3.3 Pathological Results: 3 months histomorphology measurement results are as follows: 1). the area of internal elastic plate (IEL, mm2): no significant difference between the two groups on statistics. 2). Lumen area (LA, mm2), neogenesis endomembrane area (NA, mm2) and the rate of restenosis area are significantly different between the two groups.3.4 Pathology integration: 14 days, endothelial integral points are difference statistically; it is no significant difference between 14 days'injury integral points and three months'pathologic integral points.3.5 Detected by PCNA , when it is on the 3rd month, positive expression of B group is in cattered distribution, and positive expression of A group is significantly more than Group B (P <0.05).3.6 On the 3rd months, apoptotic cells in Group B are more than Group A, with statistical difference. (P <0.05).3.7 Electron microscopy:1) Scanning electron microscopy: 14 days, endothelial cells did not cover the stents completely in Group A, suggesting the stents did not implement re-endothelialization, while in Group B, endothelial cells covered the stent basically with disorder. 3 months, both groups were re-endothelialization.2) Transmission electron microscopy: 14 days, smooth muscle cells were observed by transmission electron microscope and found that Group A had secreted SMC mostly, round cells in general, and many cell organelles, implying a strong proliferative capacity. And Group B-type had contractive SMC mostly, spindle cells in general, and less cell organelles, implying a poor proliferative capacity.Conclusion1 Human umbilical cord blood contains EPCs, in a certain culture conditions, which can differentiate to the endothelial-like cells;2 A20 gene can transfect EPCs successfully;3 Through the way of coupling with antibody,transgenic endothelial cell seeded stent can be developed;4 Vascular stent transfected by A20 gene can prevent vascular restenosis effectively.