| Objectivep21( cipl/waf-l/sidl/cap20) is one of important number of GDI protein family that can associate with cyclin-cdk complexes. Cell growth is corrected through the ability of p21 to inhibit progression through the Gl phase of the cell cycle. In this study ,our aim was to investigate expression of p21 in experimental autogenous vein grafts and analyze the relationship between p21 expression and VSMC growth. This result will direct gene transfer of p21 later and suggests that it may have application to the treatment of vascular proliferative diseases.Methods1. Animal modelAn animal model of the autogenous vein graft was established through transplanting the jugular vein into artery. It was established in 36 Wistar rats of six groups (250 50g weigh, three months old, n = 6 ) . Cutting jugular vein 0.5 cm, using 10-0 no-injure suturing, end-to-end anastomosis jugular vein into jugular artery. The vein grafts were harvested at 1 day,7 days,14 days,21 days,28 days and 42 days after operation respectively.2. Detect methods(1). Histomorphology detect: These specimens were stained by the methods of HE and Verhoeff. Morphology of tissue was observed in optics microscope. The intima thickness were measured by computer graph analysis system.(2). Immunohistochemistry: These specimens were stained with antibody of PCNA and p21, using techique of irnmunohistochemistry. These slide was observed in 400 x optics microscope and calculated the positive cells of PCNA andP21.( 3). RT-PCR analysis: With the technic of RT-PCR, all these specimens which got from different phase was extracted, degenerated, extended, amplifi-cated and electrophoresis in order to get the level of p21.3. Statistical methodsThe result was analyzed wih computer and by stasistics, statistical significance was defined at P <0.05.ResultsThe obvious neointima is seen at 14 days and the intima is fully formed by 28 days.In normal veins and arteries, p21vis not detected by tissue immunostaining or by RT-PCR. One day after vein grafts,p21 protein is observed in about 4. 6% of nitimal cells. At 7 days ,p21 protein was detected in a significant percentage of intimal SMCs. The expression of p21 protein peaks at 21 days (46. 2 å¤5. 3% ,n = 6) and decreased gradually. At 42 daiys,p21 expression is only present in 3. 2% of intimal cells. p21 mRNA is detected by technic of RT-PCR. The tendency is similar to the expression of p2l protein.Following vein grafts, SMC proliferate within the intima, which is show by PCNA expression . Intimal SMC proliferation peaks at 7 days, with 46. 3% of cells dividing and declines to 28. 2% by 14 days when PCNA expression is particularly observed in intima. After 42 days, PCNA expression decreased and the positive cell percentages are only 6. 6%.DiscussionIt is now well established that cyclins play a positive role in promoting cell cycle transitions via their ability to associate with and activate their cognate cyc-lin-dependent kinates(Cdks). Cdk2 associates with cyclinsA,D and E and has been implicated in the control of with the Gl to S phase transition in mammals. A novel 21 kDa Cdk-interacting protein,designated Cipl,has been identified.p21 is a potent, tight-binding inhibitor of Cdks and can onhibit the phosphoryla-tion of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin Dl -Cdk4 and cyclin D2-Cdk4 complexes.PCNA positive cells increased rapidly 1 day after vein grafts and peaked at 7 days. The p21 expression (protein and mRNA) were detected 24 hours after grafting and increased gradually. When p21 protein expression reach to a higher level at 7 days,the PCNA positive cell percentages bagan to decreased rapidly. p21 expression peaked at 21 days when PCNA proteins was present at less regions , which suggest that there is some relationship between p21 and PCNA.We have analyzed the role of the p21 cyclin-dependent kinase inhibitor on vascular cell growth in vivo. Endogenous p21 is expressed at increased levels 1-21 days after vein grafts and is associated with a decline in intima...
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