Analysis Of Gene Expression In The Endothelial Cell Of Human Coronary Artery And Aorta

IntroductionAtherosclerosis (AS) is the most common and the most important kind of atherosclerosis, because itsbasic damage starts from patchy thickening of the arterial intima locally, it’s also called atheroscleroticplaques. Atherosclerosis exists with high incidence,with a growing trend in recent years. It is a complexmultifactorial disease, its pathogenesis and the exact cause is not fully understood. Arterial endothelial celllayer can apperceive stress sensing blood pressure, inflammatory signals, hormones and other informationthrough the release of the active substance, react to this information, maintain vascular wall tension,bloodflow,barrier wall repair and inflammation angiogenesis and other functions.As the beginning part ofatherosclerosis,endothelial dysfunction or injury play an important role in atherosclerosis. The proximalsegment of coronary atherosclerosis may be associated with endothelial heterogeneity.Environmentalfactors may affect long-term endothelial cell expression of different genes in different parts.Currently, studyof different types of atherosclerosis and vascular endothelial basement mainly emphasis on the expressionof specific genes, studies of changing the overall gene expression are rare.Gene chips can be analyzed inparallel with a small sample of thousands of genes simultaneously in a single experiment,studying theexpression and function from the angle of the overall macroscopic objects graduate gene. Currently, genechip technology has been widely used in gene sequencing, gene expression profiling, gene mutation andpolymorphism analysis, discovery of new genes, drug screening, diagnosis and typing and so on. Thispaper uses gene chip to analysis gene expression profiling of coronary artery endothelial cells and aorticendothelial cell, screens out gene expressing differences, and explores the related functions and signalingpathways.ObjectiveUsing gene chip to analysis gene expression profiling of coronary artery endothelial cells and aorticendothelial cell, screens out gene expressing differences, and explores the related functions and signalingpathways MethodTotal RNA was extracted from coronary artery endothelial cells (HCAEC) and aortic endothelial cells(HAEC) strains, hybridization probe was prepared and purified by reverse transcription, using Agilent’shuman genome oligonucleotide microarray screening out differentially expressed genes, and then using theDAVID functional Notes bioinformatics microarray analysis system for P <0.05and greater than2-folddifference in gene expression for gene enrichment analysis and real-time quantitative PCR, immunoblotting(Western blot) partially screened gene expression validation. Building recombinant plasmid of EDN1,OCLN overexpression and stably transfected into HCAEC by liposome and screened with G418, verifiedby Western blot method to detect the implications for the rear portion of the gene expression. Then makeuse of MTT assay growth curve,test cell cycle by flow cytometry, explore impact of EDN1, OCLN on cellproliferation.ResultCompared with aortic endothelial cells, coronary artery endothelial cells express more than twice thedifference, P value less than0.05points of difference have2435, of which1,198genes were up regulated,1237genes were down regulated. By GO classification Visible differences in expression of genes involvedin cell proliferation, differentiation, adhesion, multiple genes and signaling pathways involved ininflammation, lipid metabolism, immune response, host defense, stress response, ion transport as well asangiogenesis and apoptosis; Pathways analysis of the gene can be obtained more signaling pathwaysinvolved, mainly related cancer pathways between cytokine receptor interaction, MAPK signalingpathways, adhesion molecules, cytoskeletal regulation, endocytosis, ECM receptor interaction, Jak-STATsignaling pathway and so on. Validation part gene consistent with the results of gene chip by real-timequantitative PCR. Successfully constructed EDN1, OCLN overexpression vector and stably transfected intoHCAEC, and at the protein level, Western blot showed that the gene expression level increased. MTTresults showed that HCAEC growth inhibition after over-expression of gene, flow cytometry display S-phase cells decreased,it may inhibit the cell proliferation by cell cycle.ConclusionsApplication of Gene Expression Profiling successfully screens out the differences coronary and aorticendothelial cell gene expression in endothelial cells.By biological information analysis,it mainly involvescell proliferation, differentiation, adhesion, inflammation, lipid metabolism, immune response, host defense, stress multiple genes and signaling pathways reactions, ion transport as well as angiogenesis and apoptosis,it may bring new ideas for study of the pathogenesis of coronary atherosclerosis and its prevention.Thereare affect on proliferation of coronary artery endothelial cell after gene EDN1, OCLN overexpression,itmay be associated with cell cycle arrest.