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目的 :血管平均滑肌细胞的凋亡在球囊损伤后 3 0分钟内即已经开始 ,用基因转移的方法加速这一过程是控制再狭窄的途径之一。选择野生型p5 3作为目的基因 ,观察其对血管平滑肌细胞生长及凋亡的影响。方法 :构建了野生型p5 3基因重组反转录病毒载体pLXSN -p5 3。重组载体转染培养的大鼠主动脉平滑肌细胞 ,测定转染效率和p5 3基因的表达 ,用细胞存活曲线、流式细胞仪和DNA“ladder”法检测了细胞生长和凋亡状况。结果 :采用pLXSN -p5 3转染培养的大鼠血管平滑肌 ,转染效率接近 10 0 % ,转染后的宿主细胞内可检测到外源性p5 3mRNA和外源性p5 3蛋白 ,表明基因在平滑肌细胞内获得了良好的表达。用流式细胞仪检测发现在转染后 2 4小时 ,宿主细胞G1期数量明显增多 ,S期细胞数量减少 ,而且有大量的亚二倍体细胞 ,表现为G1期抑制和细胞凋亡。采用经典的DNA“ladder”法检测发现 ,宿主细胞大量凋亡。结论 :以上结果说明外源性p5 3蛋白的表达可明显促进平滑肌细胞的凋亡。
PURPOSE: Apoptosis of vascular smooth muscle cells has begun within 30 minutes of balloon injury. The use of gene transfer to accelerate this process is one of the mechanisms that control restenosis. The wild-type p5 3 was selected as the target gene to observe its effect on vascular smooth muscle cell growth and apoptosis. Methods: The wild type p5 3 gene recombinant retrovirus vector pLXSN-p5 3 was constructed. The recombinant vector was transfected into rat aortic smooth muscle cells. The transfection efficiency and the expression of p5 3 gene were determined. The cell growth and apoptosis were detected by cell survival curve, flow cytometry and DNA “ladder” method. Results: Transfection of cultured rat vascular smooth muscle with pLXSN-p5 3 resulted in a transfection efficiency close to 100%. Exogenous p5 3 mRNA and exogenous p5 3 protein were detected in the transfected host cells, Smooth muscle cells obtained good expression. Flow cytometry showed that at 24 hours after transfection, the number of G1 phase cells in host cells increased significantly, the number of S phase cells decreased, and a large number of sub-diploid cells showed G1 phase inhibition and apoptosis. Using classical DNA “ladder” test found that a large number of host cell apoptosis. Conclusion: The above results indicate that the expression of exogenous p5 3 protein can significantly promote the apoptosis of smooth muscle cells.