| Part one Effect of Adiponectin on Inhibiting Vein Graft Restenosis after Coronary Artery Bypass GraftingObjectiveCoronary artery bypass grafting is an effective surgical treatment for coronary artery disease(CAD),which is one of the main causes of disease death in human beings.Saphenous vein grafts provide the most widely used bypass conduits for its convenience of harvesting and manipulating.However,the great saphenous vein is prone to postoperative restenosis or occlusion and difficult to maintain patency due to thrombosis,intimal hyperplasia,atherosclerosis,and other mechanisms.How to prevent restenosis and maintain the patency of the great saphenous vein is of great clinical significance.Adiponectin(ADP)is a plasma hormone protein secreted by adipose tissue with antiinflammatory,anti-atherosclerosis,anti-apoptosis,and pro-angiogenesis effects.Recent studies have shown that adiponectin receptors can be expressed on the surface of endothelial cells and fibroblasts and are involved in restenosis after vein transplantation.In this chapter,a rat model of autogenous vein transplantation was established to simulate the process of human coronary artery bypass grafting,and different doses of ADP were applied to the surface of the vein grafts,aiming to explore the influence of ADP on the process of restenosis of the vein grafts after grafting.MethodsThe autogenous jugular vein of SD rats was transplanted between carotid arteries by anastomotic cuff technique to establish the rat model of autogenous vein transplantation.Two groups of different doses of ADP(2.5?g and 7.5?g)were coated with 30% Pluronic F-127 gel as drug loading material on the surface of rat vein graft,with non-treatment group(autologous transplantation only)and 30% Pluronic F-127 gel applied group as control group.The thickness of vein grafts,immunohistochemical expression of PCNA,VCAM-1 and ICAM-1 and Western blot were observed and compared between different treatment groups to evaluate the effect of ADP on graft vein.ResultsAll operations were successful in each group.On 28 days after the operation,the vein grafts collected in the control group were thickened,the wall of the tubes was stiff,and the adhesion of the surrounding tissues was obvious,while in the high-dose ADP group,the vein was not dilated significantly,and the surrounding tissues were easy to separate.From the 3rd day after surgery,the PCNA index of the intima and adventitia in the ADP treated groups was significantly lower than that in the control group and vehicle gel treated group(P<0.01),and the expression of VCAM-1 and ICAM-1 was significantly down-regulated in the ADP treated groups(P<0.01),and the results of Western blot showed the same trend.On the 28 th day after operation,the intima,media,and adventitia thickness of vein grafts in ADP treated group were lower than those in control group and glue group,and the effect of high dose ADP on inhibiting wall thickening was more obvious than that in low dose ADP group(P < 0 05).ConclusionIn this study,a rat autologous vein model was established to explore the inhibitory effect of ADP on vein grafts restenosis by locally applying different doses of ADP on the surface of the grafted vein.These results showed that adiponectin treatment could inhibit the proliferation of intima and adventitia cells,and down-regulate the expression of VCAM-1 and ICAM-1.These results suggested that ADP could reduce the hyperplasia of intima,media,and adventitia of vein grafts,and jointly down-regulate the expression of VCAM-1 and ICAM-1 proteins related to restenosis,and finally inhibit the restenosis of vein grafts.The results of this study lay the foundation for the in-depth study of the effect of ADP on the process of vascular restenosis and provide a feasible operation method for the experimental study of vascular restenosis.Part two Establishment of a Transgenic Strain in Silkworm with Adiponectin and using in Material preparation for external stentObjectiveIn order to solve the problem of venous restenosis after CABG surgery,a large number of researches focused on the mechanism of vessel restenosis and preventing vessel restenosis or reocclusion.With the rise of interdisciplinary fields such as human medicine and biomaterials,research strategies for solving some problems in clinical medicine have gradually changed.The role of ADP in preventing venous restenosis has been demonstrated in the first part of this article.In addition,external stent also play an important role in preventing vascular restenosis and maintaining vascular patency.External stent can promote the new outer membranes formation and restrict its overdistention after placing external stents around vein grafts,and exert symmetrical pressure on vessels,thus prevent late vein graft restenosis.The combined effect of ADP and external stent to inhibit bridging vein restenosis is the main problem in this study.Silk is an exceptional biomaterial due to its remarkable mechanical strength,impressive biocompatibility with skin tissues,and adjustable biodegradability.The characteristics of silk suggested that it can use as vascular materials for vascular stent applications.The transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon,which can produce mass production of useful proteins.In this chapter,we expressed ADP protein in the middle silk gland of silkworm expression system.Then established a transgene silkworm strain,by identifying amounts of ADP and which has the stable heredity because of the highest expression level.Therewith,a materials preparation model of external stent was produced by the silk from ADP transgenic silkworm,and its properties were analyzed by material analysis method.It's worthwhile to emphasize our goal that combine with the two therapeutic tools(ADP and external stent)to treat vessel restenosis,in medicine and through transgene silk in biology.MethodsThe expression vector of middle silk gland of silkworm expression system was constructed and obtained positive moth by fluorescent screening after microinjected the successful expression vector into the eggs.The analysis of SDS-PAGE and Western Blot used to detect and quantify of rh ADP protein in the transgenic silkworm,the results were used to evaluate the silkworm strain with stable heredity.Genome's extraction could identify the insertion sites of ADP gene,and the distribution of rh ADP protein in silk was detected by immunofluorescence assay.Then investigated the economic characters of cocoons,for comparing the microstructure,secondary structure and mechanical properties between rh ADP transgenic silkworm and WT.After extracted and concentrated rha DP protein,the biological activity of rha DP protein was detected by toxicity test,cell proliferation activity and anti-inflammatory effects.Using the rh ADP transgenic silkworm cocoons to prepare the external stent materials,and analytical methods of scanning electron microscopy(SEM),FTIR,mechanical properties test and water absorption were completed for assess the material properties external stent.ResultsFirstly,we successfully established the rh ADP transgenic silkworm strain and could detect the expression of rha DP protein in the transgenic silk.The result of Western Blot analysis suggested that 7mg rh ADP could be recovered from 1 g cocoons from the transgenic silkworm,and we also found the protein modification of rh ADP protein when expressed in silkworm,of which showed a polymer structure.Exogenous gene of rh ADP was a single copy and transgene integration sites located on chromosome 10.rh ADP protein was extracted and concentrated for cell assay,the results showed that the rhADP had no cytotoxicity on 3T3-L1 cells,improved the proliferation of 3T3-L1 cells,and inhibited the inflammatory response induced by LPS of raw264.7 cells.The microstructure,secondary structure and mechanical properties of rh ADP transgenic silk,and the cocoon yield were showed insignificant difference when compared between transgenic strain and WT.After we tried to make an external stent by using rh ADP transgenic silkworm cocoon as material,the SEM showed a honeycomb-like structure from longitudinal aspect,the secondary structure analysis showed that the ?-pleated sheet ratio was increased significantly,the mechanical property test showed that the fiber strength of vessels stent is 0.14 MPa,the test of water absorption showed that the water absorption values of vessels stent material increase within the immersion time limits,along with no obvious deformation,and the final weight up to 100 times when compare with initial states.ConclusionIn this paper,Bombyx mori was used as a bioreactor to express rh ADP protein with Ser-1 expression system,and then established a transgenic silkworm strain and could get a large number of rha DP proteins by with biological activity.In addition,in view of the silk has the property of material for vascular stent,a materials model of external stent using the silk from ADP transgenic silkworm was made.In a word,we explore the possibility of treating the major heart diseases of venous restenosis by the way of biomaterials. |
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