Experimental Study On The Mechanism Of OPN Antisense Nucleotide Inhibited Rat Vascular Smooth Muscle Cell Proliferation

ObjectivePercutaneous coronary intervention (PCI) is now one of widely used methods to treat coronary artery disease, but restenosis following PCI during 6 months limits its long-term benefits.The biological mechanisms are complex, involving elastic recoil in the first instance, followed by extracellular maxtrix production, smooth muscle cell proliferation and migration, and remodeling of the vessel. Therefore the hunt for efficient inhibitors of restenosis remains an ongoing challenge.Therefore, search available road to inhibit SMC proliferation and migration have important significance for preventing RS. OPN is an important functionality protein in extracellular matrix. Differential display technique demonstrate, OPN is a marker gene on VSMC phenotype transformation, it is expression determine VSMC phenotype. Many research show that OPN obviously up-regulation in vascular injury neointimal, and it could facilitate SMC and adventitial cell proliferation and migration.So it is known as an important initiating agent in vascular injury neointimal.In spite of OPN has an important role on facilitate SMC proliferation process,but specific mechanism research has not known. The aim of this study was to approach OPN antisense nucleotide on proliferation of rat vascular smooth muscle cells(VSMCs)and study its mechanism of inhibiting proliferation, and then provide rationale and experiment evidence for OPN preventing and curing RS.Methods1. Rat A10 aortic VSMCs were culturedRat A10 aortic VSMCs were purchased from ATCC. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37℃in a humid atmosphere of 5% CO2. 2. OPN antisense nucleotide transfectionTo entrust Takara company synthesis OPN antisense nucleotide, and to utilize oligofectamine reagent FuGENE6 transient transfection on VSMC.3. Experiment methodsThe effect of OPN antisense nucleotide on proliferation of VSMC was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)metabolism measuring. FCM was performed to track cell cycle progression. RT-PCR was done to detect the mRNA level of OPN antisense nucleotide, PCNA.4.Statistical analysesAll values were expressed as mean±SD. SPSS11.0 software was used for all statistical analysis. Data were analyzed using one-way analysis of variance followed by a least significant difference test (LSD) for multiple comparisons. Differences were considered significant if p<0.05.Results1. The optical density (OD) of MTT decreased significantly in VSMCs transfected with OPN antisense nucleotide compared with that in VSMCs non-transfected.The inhibition ratio of VSMC proliferation was 24%,19.6%,17% respectively after transfecting of OPN antisense nucleotide on the 24th,48th and 72th hour. OPN antisense nucleotide can significantly inhibit 10% FBS induced VSMC proliferation.2. FCM analysis indicated that the G0/G1 phase fraction ratio of the OPN antisense nucleotide group was higher than the control group, while its S-phase fraction ratio was lower than the control group, the proliferation index of the OPN antisense nucleotide group decreased significantly. The result suggested that OPN antisense nucleotide blocked VSMC cycle in G0/G1 phase, inhibited the proliferation of VSMC.3. The results of RT-PCR showed that the PCNA was decreased obviously in the OPN antisense nucleotide group compared with the control group.ConclusionIn the rat vascular smooth muscle cell, OPN antisense nucleotide inhibited the proliferation of VSMC, its mechanism maybe is OPN antisense nucleotide blocked VSMC cycle in G0/G1 phase.