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目的研究脉冲电场对血管紧张素2型受体(AT2R)基因在血管局部表达的作用,探讨AT2R基因在体转染对大鼠颈动脉球囊损伤后新生内膜增生的作用。方法大鼠颈动脉球囊损伤后,用脉冲电穿孔法介导AT2R cDNA真核表达质粒或空质粒载体在动脉局部表达,于术后3、7 d和21 d用免疫组织化学、HE染色等方法,进行AT2R在颈动脉壁中表达的变化及定量组织形态学分析。结果免疫组织化学染色结果显示:脉冲电场介导AT2R cDNA真核表达质粒表达后,3 d时可见内膜、中膜、外膜大量AT2R的棕色阳性着色,7 d时可见少量棕色阳性着色,21 d棕色阳性着色几乎消失,未转染组及空质粒转染组未见AT2R的棕色阳性着色,表明脉冲电穿孔能有效导入AT2R基因,并在动脉壁获得稳定表达。在21 d时,AT2R cDNA真核表达质粒组的内膜面积与中膜面积比显著低于未转染组和空质粒载体组[分别为(0.76±0.08)、(1.39±0.08)、(1.32±0.10),P<0.01],空质粒转染组和未转染组间无统计学差异(P>0.01)。结论脉冲电穿孔可介导AT2R基因在血管局部有效表达,AT2R在体转染可抑制球囊损伤后大鼠颈动脉平滑肌细胞增殖和新生内膜增生。
Objective To study the effect of pulsed electric field on the local expression of angiotensin Ⅱ type 2 receptor (AT2R) gene in blood vessels and to explore the effect of AT2R gene transfection on neointimal hyperplasia after carotid artery balloon injury in rats. Methods After the carotid artery balloon injury in rats, the AT2R cDNA eukaryotic expression plasmid or empty vector was induced in the artery by pulse electroporation. The expression of AT2R cDNA was detected by immunohistochemistry and HE staining on the 3rd, 7th and 21st day after operation Methods The changes of AT2R expression in the carotid artery wall and quantitative histomorphological analysis were performed. Results Immunohistochemical staining showed that a large number of AT2R intima, tunica adventitia and adventitia were brown positive staining after 3 days expression of AT2R cDNA eukaryotic expression plasmid by pulse electric field. A small amount of brown positive staining was observed on day 7 d brown positive staining almost disappeared, untransfected group and empty plasmid transfected group showed no AT2R brown positive staining, indicating that pulsed electroporation can effectively import AT2R gene, and stable expression in the arterial wall. At 21 days, the intima-to-media area ratio of AT2R cDNA eukaryotic expression plasmid group was significantly lower than that of the untransfected and empty plasmid groups [(0.76 ± 0.08), (1.39 ± 0.08, 1.32 ± 0.10), P <0.01]. There was no significant difference between empty plasmid transfected group and untransfected group (P> 0.01). Conclusion Pulsed electroporation can induce the AT2R gene expression locally in the blood vessel. Transfection with AT2R can inhibit the proliferation of rat carotid artery smooth muscle cells and neointimal hyperplasia after balloon injury.