The Effect Of Difference Glucose Environment On The Gene Expression Proifles Of Rat Cardiac Microvascular Endothelial Cell With FGL2Gene Silencing

Background and AIM:Myocardial microvascular endothelial cells (MMVECs) is the basic structure ofthe microvascular, and also is a important endocrine organ. Many pathogenic factorsof cardiovascular diseases see myocardial microvascular endothelial cells as the targetorgan. structural dysfunction of cardiac microvascular endothelial cells is manycardiovascular disease onset reasons as well. Diabetic cardiomyopathy (DC) is acommon chronic complications of diabetes, its pathogenesis is not entirely clear yet,the mechanism of how high glucose causes endothelial cell dysfunction is not fullyunderstood also. Studies have shown that cardiac microvascular endothelial cellinjury within DC may be a precipitating factor for myocardial injury. fibrinogen-likeprotein2(Fgl2) prothrombinase also named as fiber interleukin which is known ascoagulation factors have researching hot in recent years, it may be involved in thepathophysiological process of a variety of diseases. This study was designed byhigh-throughput microarray technology to observe the effect of gene expressionprofiles of different concentrations of glucose cultured rat CMVEs with Fgl2genesilencing. Explore sugar environmental and Fgl2gene at the molecular level abouthow did they effect microvascular endothelial cell function on the genetic andmolecular mechanisms, provide a new target and ideas for the treatment of diabeticcardiomyopathy.Methods:(1) Primary rat cardiac microvascular endothelial cell extraction, culture andidentification: Take a week old healthy and clean Sprague-Dawley (SD) rats, heartwas removed in sterile, Enzyme digestion method to culture primary MMVECs.digestion and transfer of culture after the primary cell monolayer fusion. Take thesecond generation of cells were identified by immunofluorescence with MMVECscharacteristic surface antigen CD31-RA and FVIII-RA.(2) To build fgl2shRNA slow virus expression vector and no-load negativecontrol by the gene silencing design principles, and transfection MMVECs. (3) Query Ensembl RGSC3.4database about expression profile information ofthe rat, The application film chip synthesis technology synthetic rat12x135K Arraymicroarray. Each chip containing26,419genes. A single gene on each chip designwith three or more probes, and to improve the detection accuracy.(4) Experimental groups: Take the third generation MMVECs. Divided into twogroups which cultured in high-glucose and normal-glucose train. Each consist of threegroups:â‘ pure culture (control group);â‘¡Empty vector transfected (GFP group);â‘¢the fgl2RNAi slow virus transfection group (viral transfection group).(5) Stably transfected72hours after transfection, RNA extraction, detection ofRNA quality; cDNA synthesis and labeling; marked probe and microarrayhybridization; image acquisition and data analysis.Results:(1)Successfully isolated and cultured primary rat cardiac microvascularendothelial cells, the purity is more than95%. Cell morphology showed a typical"cobblestone" morphology.(2) By immunofluorescence assay, cells CD31, FVIII-positive rate of90%.CD31red fluorescence distribution in the cell membrane; FVIII distribution of greenfluorescence in the cytoplasm.(3) Detected by restriction enzyme digestion and DNA sequencing fgl2shRNAlentiviral expression vector was constructed successfully. After transfection36h byfgl2shRNA lentivirus MMVECs more than90%can be observed in the developing ofgreen fluorescent protein, to prove that the virus has been successfully transfectedinto the cell.(4) Expression microarray detection, within the group of the group of high-sugarand low-sugar group there are significant differences compared to full geneexpression profiles, viral transfection group in the high-sugar, low-sugar group weresignificantly different. Statistically significant (P<0.05).Conclusion:This experiment successfully cultured primary rat cardiac microvascularendothelial cells; fgl2shRNA lentivirus vector was successfully constructed;successfully transfected MMVECs with lentivirus fgl2shRNA; High glucose group, normal-glucose MMVECs whole gene expression within each group there aresignificant differences, prove Fgl2genes by RNA interference affects multipleMMVECs gene expression; fgl2shRNA lentivirus then incubated at different glucoseconditions, MMVECs whole gene expression there are significant differences, provedifferent glucose grown cells whose gene expression is changed. Fgl2genes by RNAinterference on myocardial microvascular endothelial cells Smoc1, Scn7a, Epyc,Mal2, Hils1, Gcnt2, St6gal1, Alk, Ccl3and so on have particularly significantinfluence. Environmental impact of sugar on MMVECs particularly evident inLOC499607, Acyp2-ps1, RGD1566264these three genes. Filter out the mostsignificant of these differentially expressed genes, pointed out the direction for furtherstudy.