| BackgroundBackground Congenital heart disease is a common disease in pediatrics,with a incidence of about 2‰.The incidence of congenital heart disease among live infants can reach about 6.7‰.About 50%of critically ill children cannot receive timely treatment and die within I year old.More and more children with complex cardiovascular diseases are receiving meridian surgery to prolong life and relieve pain.Cardiopulmonary Bypass(Cardiopulmonary Bypass,CPB)is the heart pump function by simulating the venous blood lead in vitro,maintaining a method of circulating blood.With the continuous improvement of CPB technology,CPB has played a positive and beneficial role in coronary artery bypass transplantation and other difficult and complex cardiac surgery,significantly reducing the death rate and postoperative complications of cardiac surgery patients,and is an irreplaceable and important part of cardiovascular surgery.The operational characteristics of CPB technique on repeated myocardial ischemia reperfusion make it inevitable to cause myocardial ischemia reperfusion inury(MIRI)of different degrees while assisting cardiovascular surgery,leading to decreased myocardial compliance and even serious complications such as heart failure.With the invention of the cryogenic liquid cardiac arrest and clinical promotion,myocardial ischemia-reperfusion injury after CPB were improved to some extent,however,after CPB reduced myocardial compliance and systolic dysfunction caused by low ejection fraction of heart function damage phenomenon,seriously reduces the surgical curative effect,to children caused serious influence.After CPB reperfusion,myocardial injury was more serious in children than in adults.The present myocardial protection methods,such as hypothermia and cold crystal cardiac arrest fluid,are mainly designed for adults,while there are obvious differences between immature myocardium and mature myocardium in terms of tissue structure,energy generation and utilization,excitation coupling,etc.Therefore,in view of the characteristics of immature myocardium in children,we urgently seek a method to alleviate the injury of immature myocardium after CPB,in order to reduce postoperative complications in children and effectively enhance the surgical efficacyG protein-coupled al receptor kinase 2(G protein-coupled receptorkinase2,GRK2)as targets for gene therapy in animal models and in vitro cell experiment has obtained many achievements,GRK2 namely beta adrenergic receptor kinase 1(β-adrenergic receptor kinase 1,PARK1),the study found that inhibit GRK2 expression can improve the density of ischemia anoxic myocardial tissue beta AR and sensitivity,the myocardial contraction,obvious effects in myocardial ischemia-reperfusion injury.GRK2 blocker,(3-adrenergic receptor kinase inhibitor PARKct,protects the systolic function of myocardial tissue by regulating the expression of related proteins and molecules during cardiac systolic and systolic processes,and is a popular research pathway in myocardial ischemia reperfusion injury.Matthew L transfected the βARKct into human cardiomyocytes and believed that the transfection of the βARKct enhanced cardiac systolic function.Tachibana H believed that the level of βARKct was positively correlated with the improvement of cardiac function after ischemia and hypoxia.The previous experiment of our group confirmed that the transfection of PARKct into CPB pig myocardial tissue with adenovirus as the vector could inhibit the expression of GRK2,increase the density of β1-AR,and significantly enhance the cardiac function of mature myocardium.However,due to the special physiological characteristics of immature myocardium,the protective effect of PARKct on immature myocardium after CPB has not been reported.Moreover,the application of high concentration of adenovirus vector in the myocardial tissue of the injection site caused serious damage to the myocardial tissue,and adenovirus vector had potential mutagenicity.Due to the hypoplasia of immature myocardium,the local damage of adenovirus to immature myocardium is more serious than that of mature myocardium.As a biological carrier,the storage and transportation requirements of adenovirus preparations are extremely strict.We think,can develop a more effective drug carrier,using its slow-release characteristics of stability,reducing local high concentration of adenovirus for the side effects of myocardial tissue,at the same time of maximum possible drug efficacy,reduce the side effects of drugs,so as to optimize the treatment effect of drugs,at the same time,more in-depth discussβARKct myocardial protection mechanism.Lipid nanoparticles(Lipid nanoparticles,LNPs)is a kind of double layer structure of phospholipids,respectively in the inland waters and double phospholipids film coating between hydrophilic and hydrophobic drugs.Liposomes have a large drug load and relatively stable physical and chemical properties.However,common liposomes are easy to be cleared in the body,and the effective blood concentration of the drug is difficult to guarantee.Long Circulation Lipid nanoparticles(Long Circulation Lipid nanoparticles,LC-LNPs)is a kind of polymer modification to the low immunogenicity of liposome,is a new kind of drug carrier,through the chemical modification of polymers can form a layer on the surface of liposome in hydrophilic protective film,extend the liposome systemic schedule,reduce blood clearance,its stability,long-term effectiveness and efficiency in gene carrier superiority,has high biocompatibility and targeted,can effectively protect the loaded drug against various bioactive substances by the body metabolism,With the characteristics of slow release and high loading efficiency,it is an important research direction of gene carriers in the future.Objective1.βARkct-CMD-LPNs is designed to avoid the disadvantages of biological carriers and develop a stable carrier convenient for transportation and storage,so as to minimize the toxic and side effects of drugs and optimize the therapeutic effects of drugs while maximizing the biological effects of drugs.2.Confirm the protective effect of βARKct-CMD-LPNs on immature pig myocardial tissue after CPB,and preliminarily explore the protective mechanism of it on immature myocardial tissue.3.Through in vitro cell experiments,the protective mechanism of βARKct-CMD-LPNs on anoxic and reoxygenated cardiomyocytes was investigatedMethodsBy modifying and modifying common liposomes and giving play to the advantages of nanotechnology,Long Circulation Lipid nanoparticles complex,βARKct-CMD-LPNs,was developed.In view of the intricate mechanism of MIRI after CPB,a CPB model of extracorporine circulation was constructed to observe the improvement of hemodynamics,cardiac morphology and function of immature pigs after CPB by βARKct-CMD-LPNs.The mechanism of protective effect of βARKct-CMD-LPNs on immature pig myocardium after CPB was preliminarily analyzed through blood indexes and laboratory examination of cardiac tissue after CPB.Build H9C2 cardiac muscle cells in vitro ischemia hypoxia and reoxygenation model,evaluation ofβARKct-CMD-LPNs protection of H9C2 cardiac muscle cells,through to the multiple signaling pathways in parallel study,the key to explore myocardial ischemia-reperfusion injury pathways and therapeutic targets for the realization of the βARKct-CMD-LPNs molecular mechanism from cellular level to the transformation of the large animal model provides important factual basis and theoretical support.1.CMD-LPNs Long Circulation Lipid nanoparticles vector was constructed.By linking nanotechnology with gene transfection,polymer dextran and olamine were chemically modified into ordinary liposomes by membrane dialysis,and target genes were loaded to synthesize theβARKct-CMD-LPNs nano liposome complex with special physical and chemical properties.With infrared spectrum and nuclear magnetic resonance,size and particle size distribution,Zeta potential,transmission electron microscopy and other methods of the research and development of the characterization,to evaluate the performance of its biological utilization,successfully developed Long Circulation Lipid nanoparticles composite carrier,is more advantageous to loading of purpose gene,the maximum may play a biological efficacy of drugs at the same time,as far as possible to reduce the toxic effects of drugs,to optimize the drug treatment effect.2.The CPB model of immature pigs was constructed to observe the improvement effect ofβARKct-CMD-LPNs on hemodynamics and cardiac morphology and function after CPB treatment,and to confirm the protective effect of βARKct-CMD-LPNs on pig heart after CPB treatment.Through the treatment of βARKct-CMD-LPNs,the blood and histological indexes of pigs after CPB were analyzed to study the protection and improvement of hormone levels,myocardial injury degree,receptor level and energy pathway in pigs.Rt-PCR and Western blot were used to investigate the expression of βAR at gene and protein levels.To evaluate the role ofβARKct-CMD-LPNs in apoptotic pathways,inflammatory pathways,energy pathways,etc.3.In vitro H9C2 cardiomyocyte culture results showed that after ischemia,hypoxia and reoxygenation,the secretion of cardiomyocyte inflammatory factors increased and the cardiomyocyte injury was serious.In the βARKct-CMD-LPNs group,the secretion of cardiomyocyte inflammatory factors was less and the degree of cardiomyocyte injury was less.Western blot results showed that compared with the normal control group,the expression of apoptosis indicators Caspase3 and p-Foxol in the ischemic hypoxic reoxygenation group was significantly increased in the vector control group,ischemic hypoxic reoxygenation group,and the expression of Caspase3 and p-fbxo1 in the,βARKct-CMD-LPNs group was down-regulated compared with the above two groups.Compared with the normal control group,LC3-II and Foxol expressions related to autophagy of myocardial cells in the ischemic hypoxic reoxygenation group were significantly increased in the carrier control group and the ischemic hypoxic reoxygenation group,andLC3-II and Foxo1 expressions in the PARKct-CMD-LPNs group were down-regulated compared with the above two groups.Conclusion:1.The excellent physicochemical properties and serum stability of the βARKct-CMD-LPNs nanoliposome complex are conducive to the loading of target genes,which can maximize the biological effects of drugs and minimize the toxic effects of drugs,so as to optimize the therapeutic effects of drugs.The successful development of the compound provides a novel idea of gene liposome association for drug loading,and its clinical application prospect is broad.2,βARKct-CMD-LPNs preventive application,can significantly improve cardiac function after CPB naive pig the indicators,and improve the diastolic function of myocardial tissue,reducing the excessive apoptosis occurred,on immature myocardial cell morphology and function of mitochondrial protection effect is remarkable,at the same time,improve the density of β1-AR and sensitivity,by protecting adrenergic pathway,displays the formidable myocardial protection.3.The βARKct-CMD-LPNs complex has a significant protective effect on hypoxia and reoxygenation of rat derived cardiomyocytes H9C2,and may play a protective role in immature myocardium by regulating calcium ion pathway,apoptosis,autophagy signaling pathway and other ways.Foxol positively regulates GRK2 transcription,and the βARKct-CMD-LPNs complex may play a protective role in cardiomyocytes through this regulatory role. |
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