The Mechanism Of BMSCs Modified With ENOS/F92A-Cav1 Gene On Rat Pulmonary Arterial Hypertension Treatment By KLF4 Pathway

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THE IMPACT OF ENOS/F92A-CAV1 GENE ON NO SYNTHESIS AND ANGIOGENESIS ABILITY OF BONE MARROW MESENCHYMAL STEM CELLSObjective NO widely participates a variety of physiological and pathological processes of the body as a new signaling molecule neurotransmitters and regulatory factors.As an important vasodilator active substance,NO has important therapeutic significance to endothelial dysfunction cardiovascular disease.Intravascular NO mainly synthesizes by endothelial cells with endothelial nitric oxide synthase,and caveolin is important membrane proteins in negative regulation of endothelial nitric oxide synthase.The study found the most important site of negative regulation caveolin to nitric oxide synthase is on the ninety-second amino acid,and after the caveolin ninety-second amino acids mutated to phenylalanine,mutant caveolin-1(F92A-Caveolin-1)lost this role in the negative regulation.So F92A-Caveolin-1 gene becomes the new site of action to regulate endothelial dysfunction.In endothelial cells induced environment,BMSCs can differentiate into endothelial cells,and can form vascular-like structures on the Matrigel matrix gel.Pulmonary arterial hypertension(PAH)is a kind of fatal disease which characterized by increased vascular resistance caused by progressive lung microvascular occlusion.Pathological characteristics of PAH are vascular endothelial damage,vascular remodeling and loss of arterioles.The study will construct lentiviral vector to explore the impact that the ability to synthesize NO e NOS / F92A-Cav1 gene on bone marrow mesenchymal stemcells(BMSCs)as well as angiogenesis capabilities.and hope to alleviate PAH progression.Methods In this study,we isolate and cultivante BMSCs from rat thigh and shank bone marrow by Ficoll(1.077g/ml)density gradient centrifugation method,and use flow cytometry for phaenotype identification of BMSCs.According to Lipofectamin 2000 transfection agent,transfect target genes and package plasmid to 293 T cells then concentrate and depurate lentiviral grains.The BMSCs of exponential growth phase are randomly divided into 6 group: blank group(BMSCs only),Negative control group(lentiviral grains infectet with empty vector p LVX-m CMV-Zs Green),Cav1 group(lentiviral grains infectet with LV-Cav1),F92A-Cav1 group(lentiviral grains infectet with LV-F92A-Cav1),e NOS group(lentiviral grains infectet with LV-e NOS),e NOS/F92A-Cav1 group(lentiviral grains infectet with LV-e NOS and LV-F92A-Cav1).5 days after infection,observe the expression of e NOS and Cav1 protein of each group with westong-blot method;detect the content of NO in supernatant culture medium of cells with griess method;observe the inf Iuence with the activity of BMSCs cells from generated NO with cck-8 method;observe angiogenesis ability of BMSCs of each group by angiogenesis experiment.Results Concentrated and purified lentiviral titer is 1×108TU/ml;Observed under an inverted fluorescence microscope,the efficiency of Lentiviral infectet with BMSCs is more than 85% in each groups;The results of weston-blot test show,e NOS protein expression is significantly increased in e NOS and e NOS/F92A-Cav1 group,the expression of Cav1 protein increased in Cav1,F92A-Cav1 and e NOS/F92A-Cav1 group;The content of Nitrite which is terminally stable product of NO is the highest in e NOS/F92A-Cav1 group(p<0.01)compared with the control group.The study found,as nitrogen radicals,a higher NO does not affect the activity of BMSCs cells in each group(p> 0.05);angiogenesis experiments found that the angiogenesis ability of BMSCs is the strongest in e NOS/F92A-Cav1 group.Conclusions Conclusion3: Lentiviral particles of LV-e NOS and LV-F92A-Cav1 can efficiently infect BMSCs,infection efficiency is over 90%,Detect target protein expression of masculine by weston-blot method.Compared with the control group,BMSCs Coinfectioned lentiviral particles of LV-e NOS and LV-F92A-Cav1 have the strongest ability of NO synthesis and angiogenesis strongest ability.Part ? THE MECHANISM OF BMSCS MODIFIED WITH ENOS/F92A-CAV1 GENE ON RAT PULMONARY ARTERIAL HYPERTENSION TREATMENTObjective Pulmonary arterial hypertension(PAH)is a kind of fatal disease which characterized by increased vascular resistance caused by progressive lung microvascular occlusion.Pathological characteristics of PAH are vascular endothelial damage,vascular remodeling and loss of arterioles.In recent years,although the understanding and treatment in pulmonary arterial hypertension has made considerable progress,the prognosis of PAH patients is still not very good.Traditional surgery and medication can only temporarily decrease pulmonary artery pressure,but they can't improve the situation of endothelial cell damage and loss of arterioles.Therefore,the ability to alleviate disease progression is limited,which leads to poorer curative effect in advanced patients.Thus,stem cells and gene therapy have become new research hotspots of PAH.Caveolin(Caveolin-1)is an important membrane protein as a negative regulator of endothelial nitric oxide synthase(e NOS).But after the caveolin ninety-second amino acids mutated to phenylalanine,mutant caveolin-1(F92A-Caveolin-1)lost this role in the negative regulation.It is found in vitro study that after BMSCs modified by e NOS can produce more NO without killing BMSCs cells,BMSCs infected with a variety of lentivirus still have the ability to form blood vessels,manifested as BMSCs of e NOS/F92A-Cav1 group have a stronger ability of producing more NO and angiopoiesis.Therefore,this study aimed to explore the specific molecular mechanism that BMSCs modified by e NOS/F92A-Cav1 reduce pulmonary artery pressure in rats,establish the scientific theoretical basis for the research of PAH therapy of gene-modified BMSCs-based in the future.Methods First,establish pulmonary hypertension model in Wistar rats by monocrotaline;second,two weeks after the establishment of PAH,electrophysiolograph detects and records pulmonary artery pressure curve of rats,calculate the average pulmonary arterial pressure(MPAP),weigh the right ventricle(RV)and the left ventricular(LV)and interventricular septum(S)of rats,calculate the pachynsis exponent of right ventricular(RV/(LV+S));stained slides with hematoxylin-eosin method(HE),measuringintima thickness(MT)and vascular outer diameter(ED)of lung Small artery,calculate the lumen area(VA),the total area of the blood vessel(TAA),MT/ED(MT%)and VA/TAA(VA%)of pulmonary artery.Then based on the results of in vivo tests,the experimental animals were randomly divided into 5 groups: normal control group(normal rats),PAH group(normal saline treatment of intravenous injection),the negative control group(BMSCs treatment of injected with empty vector infection),e NOS group(BMSCs treatment of injected with LV-e NOS infection),e NOS/F92A-Cav1 group(BMSCs treatment of injected with LV-e NOS and LV-F92A-Cav1 co-infection).experimental animal model 2W later,transplante lentivirus infection BMSCs cells in each group of 2 × 106 / ml into the body of the corresponding group of experimental animals with the vena caudalis injection method,2w times for a course,two courses of treatment,observe survival and other general conditions of rats after two courses;finally,electrophysiolograph detects and records pulmonary artery pressure curve of rats,calculate the average pulmonary arterial pressure(MPAP),weigh the right ventricle(RV)and the left ventricular(LV)and interventricular septum(S)of rats,calculate the pachynsis exponent of right ventricular(RV/(LV+S));stained slides with hematoxylin-eosin method(HE),measuring intima thickness(MT)and vascular outer diameter(ED)of lung Small artery,calculate the lumen area(VA),the total area of the blood vessel(TAA),MT/ED(MT%)and VA/TAA(VA%)of pulmonary artery;Observe the expression level of KLF4,e NOS,PGIS and ET1 m RNA of pulmonary tissue with PCR method;research the expression of KLF4,e NOS,PGIS,ET1 protein with western-blot method.Results Compared with PAH rats model,BMSCs cells given with e NOS/F92A-Cav1 loading can more effectively reduce pulmonary arterial pressure of PAH rats(p<0.01);inhibit the proliferation of pulmonary vascular smooth muscle cell;reduced right ventricular hypertrophy index(RV/(LV+S))(p<0.01)and pulmonary artery tunica media thickness index(MT%)(p<0.01),increase vascular lumen area index(VA%)(p<0.01);Realtime-PCR and western-blot study found that the expression levels of vasoconstrictor factors ET1 m RNA and protein are reduced in pulmonary tissue of rats with cells transplanted by BMSCs in each group(p<0.05,p<0.01),while the expression levels of vasodilator factors e NOS,PGIS m RNA and protein are increased(p<0.05,p<0.01),the expression levels of the transcription factors KLF4 m RNA and protein are increased,which is the most significant in e NOS / F92A-Cav1 group(p<0.01).ConclusionsThe issue successfully constructs an animal model of PAH,access to scientific research system in vivo experiments;The studies have shown that BMSCs cells loading with e NOS/F92A-Cav1 can effectively reduce the pulmonary arterial pressure,right ventricular hypertrophy index and pulmonary artery tunica media thickness index of PAH rats.increase vascular lumen area index,inhibit the proliferation of vascular smooth muscle cell;In addition,BMSCs cells loading with e NOS/F92A-Cav1 can reduce the expression levels of vasoconstrictor factors ET1 m RNA and protein,increase vasodilator factors e NOS,PGIS and the expression levels of the transcription factors KLF4 m RNA and protein(p<0.01).