SiRNA On Rat Vascular Smooth Muscle Cells The Expression Of Early Growth Response Factor-1

ObjectiveRNA interference is a normal gene expression in vivo inhibition of a specific phenomenon, it means that when the cells into the coding region and endogenous mRNA homologous double stranded RNA (dsRNA),. Exogenous dsRNA into cells generated small interfering RNA (siRNA) antisense nucleic acid chain and a variety of enzymes formed silencing complex (RNA-induced silencing complex, RISC), RISC binding and cutting with the mRNA of the role of RNA interference-mediated process. RNAi has the specificity and efficiency.Early growth response factor-1 (Egr-1) is a zinc finger transcription factor, not only with cell growth, but also involved in a variety of normal cell differentiation, apoptosis, and cardiovascular disease pathophysiology. Egr-1 in the normal vessel wall is low or no expression, while in arterial damage and upregulation of a variety of other stimuli, and to promote gene transcription and expression of vascular smooth muscle cell division, value added and intimal hyperplasia.In this study,we observed the expression of rat vascular smooth muscle cells Egr-1 both from mRNA and protein., and then siRNA transfected cells observed changes in the expression of Egr-1.Methods1.VSMC cultureThe cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), pH7.4, containing 10% fetal bovine serum (FBS), at 37℃in a humidified atmosphere of 5% CO2. The medium was changed once every two days.when Cells were passaged by washing once in phosphate buffered solution (PBS) followed by trypsinization. Subcultured strains were cultured in DMEM, pH7.4, containing 10% FBS. Subcultured strains were used between passages 3 and 8. Cell were used in trial, when growed 70%-80%.2.experiment methods(1)VSMC after 48h starvation, stimulated with 10% fetal bovine serum, collected VSMC at Oh,0.5h, 1h,2h,12h,24h,then adjust the concentration of RNA extracted, amplified after reverse transcription, for electrophoresis, EB staining, Automatic image analysis system Gel measuring Egr-1 mRNA value.(2)VSMC after 48h starvation, stimulated with 10% fetal bovine serum, collected VSMC at Oh, 1h,2h,4h,24h, using western-blot method measured Egr-1 protein value.(3)Preparation of siRNA, transfected into VSMC, stimulated with 10% fetal bovine serum 1h and 2h then measured Egr-1 mRNA and protein value.Results1.Compared with the control group (internal reference GAPDH), Egr-1mRNA content increased gradually over time, the peak in 1h then gradually decreased.2.Compared with the control group (internal referenceβ-actin), Egr-1 protein content increased gradually over time,the peak at 2h then gradually decreased.3.Compared with and the control group (no transfection of siRNA) and negative control group (transfected invalid siRNA),siRNA significantly inhibited the VSMC Egr-1 expression, Egr-1mRNA and protein were lower at the peak.Conclusion1.VSMC after starvation, stimulated with 10% fetal bovine serum,Egr-1 mRNA peaked at 1h, protein peaked at 2h.2.siRNA significantly inhibited the expression of VSMC Egr-1...