| Objective: Due to the rapid development of endovascular interventional therapy,more and more patients are receiving endovascular interventional therapy.In most clinical centers,vascular stents are used in 80% of interventional procedures.Restenosis after stent implantation((In Stent Restenosis,ISR)is still the most serious problem after arterial interventional therapy.Previous studies have shown that matrix metalloproteinases(matrix metalloproteinases,MMPs)play an important role in the process of restenosis after stent implantation,and exogenous matrix metalloproteinase inhibitors can significantly inhibit the activity of MMPs and the ability of migration and proliferation of smooth muscle cells.In this study,Z-type bare stent and drug-coated stent containing GM-6001 were used in vivo experiment and drug-coated stent degradation experiment in vitro.After non-invasive removal of metal stents from frozen specimens,the expression level of MMP-2,MMP-9,TIMP-1,TIMP-2,different phenotypes of VSMC,main collagen content,percentage of apoptotic smooth muscle cells and density of smooth muscle cells during ISR were measured.To clarify the distribution characteristics and changes of MMP activity in time and space during ISR,and to clarify the expression levels of MMPs and TIMPs,the functional evolution of VSMCs,the changes of ECM metabolism and the relationship between ECM metabolism and MMP activity.It was found that the local release of exogenous MMP inhibitors on MMP activity,VSMC quantity and ECM metabolism and regulation mechanism,which opens up new research fields and provides a new objective basis for the study of inhibition of ISR.Material and Methods: It consists of three parts.The first part is the preparation of GM6001 drug-coated stents: lactic acid and glycolic acid were dissolved in dimethyl sulfoxide in different proportions,degradation experiments in vitro,drug concentration-time curve measurement and coating screening,screening of drug-coated polylactic-glycolic acid copolymer stents prepared for animal experiments GM6001-eluting stents and GM6001-free eluting stents.The bare stent is a Z-shaped stent made of 316 L stainless steel wire.The diameter of the stent rod is 0.15 mm,the diameter of the stent is 6mm,and the length of the stent is 15 mm.The second part is animal grouping,iliac artery stent implantation and artery tissue acquisition: 42GM6001-eluting stents were randomly implanted into the left or right iliac arteries of Guangxi Bama miniature pigs,and the stents without GM6001 were implanted into the contralateral iliac arteries to form a control group.According to the time of execution(6hours,1,7,14,56,84 and 336 days,blank control),the rats were divided into 8 groups(6rats in each group).After the iliac artery was obtained,the iliac artery was soaked and embedded in OCT solution,cryopreserved in a liquid nitrogen tank,and the stent was removed in the frozen state.The thick frozen sections of 10 um were cut from the middle part of the restenotic vessels with a frozen section machine.Protein was extracted from both ends of restenosis vascular tissue for Western Blotting.The third part is specimen staining and Western blotting: staining for hematoxylin-eosin(Hematoxylin and eosin,HE(hematoxylin eosin)staining,immunohistochemical staining,Sirius red staining and terminal deoxynucleotidyl transferase d UTP labeling staining(Terminal deoxynucleotidyl transferase d UTP nick end labeling staining,TUNEL).The morphology of restenosis artery,MMP-2,MMP-9,TIMP-1,IMP-2,MYH-10(proliferative phenotype vascular smooth muscle marker),SM22 ?(contractile phenotypic vascular smooth muscle marker),apoptosis and the distribution trend of collagen in different mature states in ISR were analyzed.Results: 1.The ratio of lactic acid to glycolic acid of coated PLGA with stable drug release and release duration of more than 3 months was 70 max 30,and the diameter6 mm stents containing GM6001(carrying GM6001 5 ? g / mm2)and GM6001-free stents were made for follow-up animal experiments.2.There was no significant difference in baseline indexes such as initial body weight,blood glucose and blood lipids among the experimental artery groups(P > 0.05).All experimental animals are male,in order to exclude the protective effect of estrogen on arteriosclerosis.The animals were fed with a high-fat diet in the laboratory,which increased the probability of restenosis in the animal model.After the iliac artery of the stent was removed,the connection points at both ends of the stent were exposed under freezing condition,and tweezers were used to remove all the connection points,so that all the pillars of the stent were separated from each other and,without damaging the vascular tissue structure,the strut was pulled out longitudinally,and the Z-type stent in the vessel wall was successfully pulled out.3.HE staining showed that temporary matrix formation was found in the vascular lumen on the1 st day in the control group and on the 7th and 14 th day in the GM6001 group.Neointima could be observed in the control group from the 14 th day.The appearance of neointima in the GM6001 group was later than that in the control group on the 56 th day.Compared with the control group,the area of vascular lumen in GM6001 group increased significantly and the area of neointima decreased significantly from 7 days to 336 days.The results of immunohistochemistry showed that MMP-2,MMP-9,SM22 ?,MYH-10,TIMP-1 and TIMP-2 were mainly distributed in the intima near the internal elastic membrane,the injured part of the intima,around the stent rod or on the intimal surface.In both groups the expression of MMP-2 and MMP-9 reached the peak but GM6001-eluting stent significantly inhibited the expression of MMP-2 and MMP-9 in vascular media and neointima especially around the stent rod.The expression of TIMP-1,TIMP-2 and MYH-10 in GM6001 group was also significantly lower than that in control group.There was no significant difference in the expression of SM22 ? between the two groups.The results of TUNEL showed that the percentage and density of apoptotic cells in neointima decreased with time.The percentage of apoptosis and cell density in GM6001 group were significantly lower than those in control group.The results of Sirius red staining showed that the content of mature collagen in neointima increased with time.In the control group,the content of immature collagen gradually decreased from 56 days,but in the GM6001 group,the content of immature collagen reached the maximum at 84 days and then decreased.The contents of immature collagen and mature collagen in GM6001 group were significantly lower than those in control group.Conclusion: Our research results show that: 1.GM6001-eluting stents can inhibit the expression of MMP-2 and MMP-9 in media and neointima.2.GM6001-eluting stents reduced the number of VSMCs in neointima and significantly reduced the collagen content of ECM in neointima.3.GM6001-eluting stent can effectively inhibit neointimal hyperplasia and increase the area of arterial lumen.Our study provides a theoretical basis for the regulation of MMP activity by local release of exogenous MMPI during ISR and the inhibition of stent restenosis by VSMCs and ECM metabolism. |
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