反义凝血酶受体和p21双基因共表达对损伤的大鼠颈总动脉的影响

The Effect of Both p21 and Antisense Thrombin Receptor Genes Co-Expression on the Injured Rat Common Carotid Artery

目的 :观察反义凝血酶受体 (ATR)和 p2 1双基因治疗对WKY大鼠颈总动脉损伤后新生内膜的抑制效果 ,探寻基因治疗再狭窄的有效途径。 方法 :将 42只雄性WKY大鼠随机分成基因治疗组 (导入rAAV/AP组 ,n =18)、载体对照组 (导入rAAV/GFP组 ,n =18)和正常对照组 (n =6 )。构建含有ATR和 p2 1双基因及含有绿色荧光蛋白 (GFP)基因的腺病毒伴随病毒(AAV)载体pSNAV/AP和 pSNAV/GFP ,用重组单纯疱疹病毒 (rHSV )生产体系包装重组腺病毒伴随病毒 (rAAV ) :rAAV/AP和rAAV/GFP。将rAAV/AP或rAAV /GFP导入拉伤的WKY大鼠的颈总动脉。分别于术后 2、 4、 6周取材 ,用半定量—逆转录多聚酶链反应和免疫组织化学方法检测凝血酶受体 (TR)和 p2 1在动脉壁中的表达。图像分析和统计学检验双基因的疗效。 结果 :逆转录多聚酶链反应和免疫组织化学结果显示外源基因已导入血管壁的新生内膜和中膜并获得稳定表达。图像和统计学分析说明 :基因治疗后 2、 4和 6周 ,双基因治疗组 ( p2 1和ATR)的颈总动脉内膜厚度分别较载体对照组组内膜减少 5 9 2 %、 5 8 7%、 6 4 1%;中膜厚度分别减低 7 8%、 10 8%、 8 3%;中膜细胞密度减低 30 0 %、2 9 4 %、 2 6 2 %。 结论 :ATR和p2

Objectives:To study the inhibiting effect of antisense thrombin receptor gene(ATR) and p21 gene therapy on the intimal hyperplasia of injured common carotid artery of WKY rat,and to explore more effective method of gene therapy for restenosis. Methods:Plasmids pSNAV/AP and pSNAV/GFP,containing both expression cassette of ATR and p21 genes and of GFP gene,were constructed respectively.Recombinant adeno-associated virus(rAAV),rAAV/GFP and rAAV/AP,were packaged by using recombinant herpes simplex virus packaging system.The WKY rats were sacrificed at the end of 2,4 and 6 weeks after rAAV/AP and rAAV/GFP delivered into the balloon-injured carotid arteries.RT-PCR and immunostaining were performed to examine the expression of thrombin receptor(TR) and p21.To examine the effect of gene therapy,morphometric and statistical analyses were performed. Results:The results of RT-PCR and immunostaining indicated that the transgenes had already expressed in the injured vascular wall,and the results of morphometric and statistical analyses demonstrated that the intimal thickness was decreased by 59.2%,58.7% and 64.1%,while the medial thickness was induced by 7.8%,10.8% and 8.3%,and the cell density of media were decreased by 30.0%,29.4% and 26.2% respectively at the end of 2,4 and 6 weeks after the gene therapy. Conclusion:The proliferation of intimal and medial cells of the injured carotid arteries can be inhibited markedly by both of ATR and p21 gene co-expression.