| Background :It is found that the final effect of angioplasty is significantly limited by the high rate of restenosis,which is based on the abnormal proliferation and migration of vascular smooth muscle cells(VSMCs). After vascular injury,VSMCs are stimulated to divide in response to mitogens,and they exit the G\ phase and enter the S phase.Progression through GI and entry into the S phase is regulated by the formation and activation of cyclin/cyclin-dependent kinase(CDK) complexes.p27 is one of the key CDK inhibitor in cell cycle and inhibits the cell proliferation through inhibition of pRb phosphorylation by supperssion of the activity of several cyclin/CDK complexes.At present,it is still unknown whether the overexpression of p27 through gene recombinant techique can inhibit the VSMCs proliferation and migration.In this study,p27 gene is transferred into the cultured rat VSMCs mediated by recombinant adenovirus vector and its effects on the proliferation and migration of VSMCs are detected. Secondly, It is explored that the regulative mechanism of p27 expression in transcription level through investigating the change of p27 gene promoter activity.Objective:The purposes of present study are as follows: l)to construct the adenoviral vector containing p27 gene;2)to investigate the effects of overexprission of p27 on the proliferation and migration of VSMCs induced by platelet-derived growth factor-BB(PDGF-BB)in vitro and explore its mechanism;3)to investigate the change of p27 gene promoter activity ofVSMCs pretreated with PDGF-BB and anti-proliferative drug SDZ RAD.Method: The VSMCs,gotten from the aorta of rat,were cultured by routine method according to previous studies.Recombinant adenoviral vector,AdCMV-p27,containing human p27 gene was constructed by site special recombination,and it was used to transfer p27 gene into VSMCs.The VSMC expressed p27 gene was used as a cell model to study the function on the cells in vitro.The rate of p27 expression in VSMC was analyzed by flow cytometry.The protein expressions of p27,p21,proliferating cell nuclear antigen(PCNA),CDK2,Cyclin E,c-fos,c-myc and connexin43 were detected by immunocytochemistry method.The mRNA expressions of p27,p21,PCNA,c-fos and c-myc were semiquantitivly determined by RT-PCR.To detect the change of VSMC proliferation features,flow cytometry was used to analyse the cell cycle of VSMCs,and the assays including 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenytetrazolium bromide(MTT) and [3H] thymidine incorporation were used to determine the proliferation of VSMCs,respectively.The modified Boyden's chamber method was used to test the number of VSMC migration. The Chloramphenicol acetyltransferase enzyme-linked immunosorbent assay (CAT ELISA) method was used to investigate the effect of PDGF-BB and SDZ RAD on the p27 promoter activity in VSMCs.Results: The expression rate of p27 in VSMCs was significantly increasing after transfected by AdCMV-p27 with the time,and the peak value detected by flow cytometry was about 85.81% at 24th hour.The results of immunocytochemistry and RT-PCR showed that the protein translation and mRNA of p27 were obviously increased in transfected VSMCs. When the expression of p27 is at the peak value,the ratio of S and G2-M phase stimulated by PDGF-BB was reduced from 42.93% to 28.28%(p<0.05) in transfected cells compared with non-transfected cells.The A values of MTT and [3H] thymidine incorporation induced by PDGF-BB were reduced by 76% and 35.7%,respectively(transfected cells vs non-transfected cells, p<0.01).Thenumber of VSMCs migration induced by PDGF-BB was reduced to 54.6% at 24 hour after transfecting p27(p<0.05).Compared with the PDGF-BB stimulated group, the postive expressions of p27 and p21 protein after transfected p27 were significantly increased to 33.91% and 50.32%,respectively.And that the mRNA expression intensities of them were also obviously increased to 2.97 and 2.3(p<0.01).Although the postive grain expression ratio of CDK,PCNA,Cyclin E,c-fos,c-myc and connexin43 was remarkably increa...
|
PDF Full Text Request