重组腺病毒介导的人一氧化氮合酶基因转染对大鼠颈总动脉球囊损伤后新生内膜的影响

Effects of adenovirus-mediated endothelial nitric oxide synthase gene transfer on neointimal proliferation of rat carotid artery after balloon injury

目的 :研究重组腺病毒介导的人内皮型一氧化氮合酶基因 (eNOS)表达生成的一氧化氮合酶 (NOS)对球囊损伤后大鼠颈总动脉新生内膜的抑制作用。方法 :在 2 93细胞内扩增、纯化Ad LacZ和Ad eNOS ,鉴定其是否携带有LacZ和eNOS基因。建立大鼠颈总动脉球囊损伤模型后 ,将磷酸缓冲液 (PBS)、Ad LacZ和Ad eNOS在体内分别转染到损伤血管段 ,以X gal染色、苏木精 伊红染色 ,免疫组化及计算机图像分析处理等方法观察转染动脉节段外源性eNOS蛋白表达及其对新生内膜的影响。结果 :重组腺病毒携带有eNOS基因 ,并且在损伤血管段得到有效表达。转染后PBS组、Ad LacZ组和Ad eNOS组的新生内膜面积分别为 (0 .187± 0 .0 18)、(0 .134± 0 .0 6 1)和 (0 .0 6 3± 0 .0 2 6 )mm 2 ,新生内膜与中膜面积比值 (I/M)分别为 1.5 76± 0 .2 73、1.342± 0 .35 7和 0 .5 6 0± 0 .16 1。与PBS组、Ad LacZ组相比Ad eNOS组无论新内膜面积 ,还是管腔狭窄程度都明显减小。结论 :腺病毒介导的eNOS基因转染能有效抑制球囊损伤后血管内膜的增生 。

Objective:To study the effects of adenovirus mediated human endothelial nitric oxide synthase gene(eNOS) transfer on neointimal proliferation of rat carotid artery after balloon injury.Methods:The recombinant viruses were amplified on confluent 293 cells and purified by discountinous CsCl gradient centrifugation. The morphology of 293 cells, X gal staining on 293 cells and PCR of eNOS gene cDNA segment were employed. After balloon injury, rats' carotid arteries were immediately transduced with phosphate buffered saline(PBS) 0.2 ml, 1.02 ×10 12 pfu/ml Ad LacZ 0.2 ml and 1.24 ×10 12 pfu/ml Ad eNOS 0.2 ml in vivo respectively. X gal staining, HE staining, immunochemistry and computer image analgsis system were used to analyze the expression of exogenous eNOS protein and its effects on neointimal proliferation.Results:The transfected 293 cells were stained blue with X gal. The eNOS cDNA was confirmed in the Ad eNOS genome by PCR. After 2 weeks, eNOS protein were confirmed by immunochemistry in Ad eNOS infected segments. In PBS?Ad LacZ infected and eNOS infected segments, the neointimal area was ( 0.187 ± 0.018 ) mm 2, ( 0.134 ± 0.061 ) mm 2 and( 0.063 ± 0.026 ) mm 2 respectively, the ratio of neointima to media was 1.576 ± 0.273 , 1.342 ± 0.357 and 0.560 ± 0.161 respectively. The neointimal area and the I/M ratio of Ad eNOS infected were significantly reduced.Conclusion:Adenovirus mediated NOS gene transfer might reduce arterial restenosis after balloon angioplasty.