| Objectives:(1)To construct the gene therapy vector,p MI1-AAVP-PDGF-B,which is able to be used by vein and mediate targeting therapeutic angiogenesis in ischemic myocardium;(2)To investigate therapeutic effect of the p MI1-AAVP-PDGF-B phage for myocardial infarction and the related mechanism;(3)To investigate whether cardiac telocytes(CTs)exist in Xenopus tropicalis(X.tropicalis)using transmission electron microscope(TEM).Methods:(1)The molecular clone technique was applied to contruct the p MI1-AAVP-PDGF-B targeting vector,which is able to display the cardiac vasculature targeting peptide MI1 on the surface of phage to mediate the myocardial vasculature targeting and also includes cis-element of adenovirus-derived vector to conduct the expression of human PDGF-B in mammalian,the p MI2-AAVP-PDGF-B vector(Non-cardiac vasculature targeting peptide control),p MI1-AAVP-PDGF-B-EGFP vector.The p MI1-AAVP-PDGF-B vector,The p MI2-AAVP-PDGF-B vector and p MI1-AAVP-PDGF-B-EGFP vector were packaged as phages and then phage concentration was determined by titration respectively.(2)The prepared p MI1-AAVP-PDGF-B phages and p MI2-AAVP-PDGF-B-EGFP phages were applied by intravenous injection into Anterior descending coronary artery ligated(LAD)rats.The heart,liver,lung,spleen,brain,kidney,muscle of the treated rat were collected in 10 min and 24 hour after injection for anti-phage inmmunofluorescence staining to investigate the tissue and organ specific targeting.(3)The prepared p MI1-AAVP-PDGF-B phages and p MI2-AAVP-PDGF-B-EGFP phages were applied by intravenous injection into LAD rats respectively.The injected hearts were collected and conducted the RT-PCR analysis for human PDGF-B and anti-EGFP inmmunofluorescence staining in 1 week after injection.(4)The p MI1-AAVP-PDGF-B phages,p MI2-AAVP-PDGF-B-EGFP phages,p MI1-AAVP phages and PBS were applied by intravenous injection into LAD rats respectively.The cardiac function in treated rats were analyzed by echocardiography in 2 weeks after injection.In 2 months after injection,the cardiac function and the physical ability analyzed by echocardiography and exercise ability index.Hematoxylin-Eosin staining was applied to evaluate the side effects of injected phages in histologyical level.The Masson's Trichrome staining and immunohistochemistry staining were applied to analyze infarct size,fibrosis,wall thickness,the vessel density and the number of survival cardiomyocytes and proliferative of cardiomyocytes in the infarct myocardium.(5)TEM ultrastructure analysis was applied to observe CTs in representative regaion,the atrium-atrial region,medium middle region and base of the X.tropicalis heart.In addition,the wound region of amputated heart was analyzed by TEM in 2 days and 8 days after apeical resection of X.tropicalis heart to investigate whether CTs is involved in regeneration of X.tropicalis myocardium.Result:(1)The sequence of the constructed p MI1-AAVP-PDGF-B vector,the p MI2-AAVP-PDGF-B vector and the p MI1-AAVP-PDGF-B-EGFP vector are confirmed by DNA sequencing.The p MI1-AAVP-PDGF-B-EGFP vector is able to expression in 293 T cell in 24 hours after transfection.The titer of prepared p MI1-AAVP-PDGF-B phages,p MI2-AAVP-PDGF-B phages and p MI1-AAVP-PDGF-B-EGFP phages is 1011pfu/m L after package.(2)The targeting ability of contructed phages for ischemic myocardium: The results of anti-phage immunofluorescence staining showed that the positive staining of p MI1-AAVP-PDGF-B phage injected LAD heart in both infracted zone and border zone in 10 minutes after injection was significantly higher than that of p MI2-AAVP-PDGF-B phage injected heart.Fourther more,the positive staining was still able to be observed in p MI1-AAVP-PDGF-B phage injected heart in infracted zone in 24 hours after injection,but it was rare in the p MI2-AAVP-PDGF-B phage injected heart.(3)In vivo expression of constructed phages: RT-PCR revealed that the human PDGF-B was expressed in infracted zone in p MI1-AAVP-PDGF-B phage injected LAD heart in 1 week after p MI1-AAVP-PDGF-B phage injection.In addition,RT-PCR and anti-EGFP immunohistochemistry staining documented that EGFP was expressed in infracted zone in p MI1-AAVP-PDGF-B phage injected LAD heart in 1 week after p MI1-AAVP-PDGF-B phage injection.(4)The therapeutic effects of the p MI1-AAVP-PDGF-B phage injection for myocardial infarction:(1)For cardiac function evaluation,the ejection fraction and shortening fraction of p MI1-AAVP-PDGF-B phage injected LAD heart in 2 weeks after injection were significnantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBS-injected heart respectively(p<0.05).However,there was no significance difference for the left ventricular end-diastolic diameter and left ventricular end-systolic diameter,the left ventricular end-diastolic volume and left ventricular end-systolic volume in among p MI1-AAVP-PDGF-B phage-,p MI2-AAVP-PDGF-B phage-,p MI1-AAVPphage-and PBS-injected heart(p>0.05).In 2 months after injection,the ejection fraction and shortening fraction of p MI1-AAVP-PDGF-B phage injected LAD heart were significnantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBS-injected heart respectively(p<0.05).The left ventricular end-systolic diameter and left ventricular end-systolic volume of the p MI1-AAVP-PDGF-B phage injected LAD heart were significnantly smaller than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBSinjected heart respectively(p<0.05).While there was no significance difference for left ventricular end-diastolic diameter,left ventricular end-diastolic volume in among p MI1-AAVP-PDGF-B phage-,p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBSinjected heart(p>0.05);(2)For the physical ability,the forelimb grip strength of the p MI1-AAVP-PDGF-B phage injected LAD heart group in 2 months after injection was significantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBSinjected group respectively(p<0.05),while there was no significance in endurance,rearing count and treadmill running in among p MI1-AAVP-PDGF-B phage-,p MI2-AAVP-PDGF-B phage-,p MI1-AAVPphage-and PBS-injected heart(p>0.05);(3)For the infarct size,the infarct size of the p MI1-AAVP-PDGF-B phage injected LAD heart in 2 months after injection was significantly smaller than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBS-injected group respectively(p<0.05);(4)For the index of the left ventricular remodeling,the smallest wall thickness in the infarct zone and the wall thickness in the border zone of p MI1-AAVP-PDGF-B phage injected LAD heart in 2 months after injection were significantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBS-injected group respectively(p<0.05);(5)For cardiac angiogenesis,the blood vessel density of the p MI1-AAVP-PDGF-B phage injected LAD heart in 2 months after injection in both infarct zone and border zone was significantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBSinjected group respectively(p<0.05);(6)For the cardiomyocyte survival and proliferation,the density of survival cardiomyocyte and proliferative cardiomycyte of the p MI1-AAVP-PDGF-B phage injected LAD heart in 2 months after injection in the infarct area and the border zone were significantly higher than those of p MI2-AAVP-PDGF-B phage-,p MI1-AAVP phage-and PBS-injected group respectively(p<0.05).(5)The results of TEM demonstrated that the cardiac telocytes exist in the X.tropicalis myocardium.CTs were mainly twined around the surface of cardiomyocyte trabeculae and were linked together via the ends of telopodes,producing a three-dimensional network of CTs in the X.tropicalis myocardium.In addition,CTs could communicate with other cells in myocardium(such as cardiomyocytes)through the cell body and telopode secreted the microvesicles and coated vesicles.In 2 days after apeical resection of X.tropicalis heart,there were many scattered red blood cells and inflammatory cells in the wound.Some of telopodes in the wound was found in edema-like morphorlogy.While some clot structures were found in the extracellular space between cardiomyocytes and CTs.In addition,the wound area contained some network structures that consisted of disorganized telopodes and extracellular matrix tissues but lacked cardiomyocytes.In 8 days after after apeical resection of X.tropicalis heart,some injured muscle fibers regenerated via a novel muscle fiber characterized by an irregular muscle fibril arrangement.In addition,the density of mitochondria was significantly higher in the regenerated muscle fibers than in the muscle fibers of the undamaged myocardium.Conclusion:(1)p MI1-AAVP-PDGF-B vector which is able to be used by vein and mediate targeting therapeutic angiogenesis in ischemic myocardium is constructed;(2)Intravenous injection of p MI1-AAVP-PDGF-B phage in LAD rat is able to improve cardiac function,decrease infarct size and improve left ventricular remodeling significantly.The therapeutic effects is able to maintained at least 2 months.The underlined mechanism of p MI1-AAVP-PDGF-B phage for myocardial infarction is attributed to promote angiogenesis in the infarct zone and border zone,decrease cardiac fibrosis,improve left ventricular remodeling,and promote survival and proliferation of cardiomyocyte in the infarct zone;(3)Cardiac telocytes exist in the adult Xenopus tropicalis heart.The regeneration of CTs and their networks might be a critical step for initiation of the regeneration of damaged myocardium. |
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