蛋白涂层支架携带质粒介导的一氧化氮合酶基因预防冠状动脉球囊扩张后再狭窄的实验研究

The local transfer of plasmid-mediated human inducible nitric oxide synthase gene with protein-coated metallic stents inhibits intimal hyperplasia following coronary angioplasty in mini-swine model

目的 为评价蛋白涂层金属支架携带质粒介导的诱导性一氧化氮合酶 (iNOS)基因局部转染血管壁 ,预防冠状动脉内血管成形术后再狭窄的效果。方法 金属支架涂层为胶联明胶制成。载体为去内毒素纯化pcDNA3。采用标准球囊导管技术 ,将吸附有质粒介导的人肝脏的iNOS基因(pcDNA3hepiNOS)涂层支架置入小型猪 (n =9)冠状动脉前降支中段 ,以相同方法置入单纯蛋白涂层支架做为对照组 (n =9) ,支架与血管直径之比为 1.1～ 1.3:1。结果 在支架置入后 7d ,RT PCR检测和免疫组织化学染色 ,证实在pcDNA3hepiNOS转染的血管段有iNOSmRNA的表达和iNOS蛋白生成 ,而远离器官则无基因的表达。 3个月时冠状动脉造影显示 :转染pcDNA3hepiNOS组 (n =5 )无再狭窄发生 ,而对照组均发生显著的再狭窄。组织病理学形态分析结果显示 :pcDNA3hepiNOS组新生内膜面积 (1.7± 0 .8)mm2 、平均百分狭窄面积 (2 6 .5± 7.5 ) %、平均管腔狭窄百分数 (4 1.2± 16 .5 ) % ,均较对照组小 ,对照组分别为 (2 .8± 0 .8)mm2 ,P <0 .0 5 ;(94.2± 14.3) % ,P <0 .0 0 1;(88.0± 16 .6 ) % ,P <0 .0 0 1;比较内膜面积 /中膜面积比值 (I/M)治疗组较对照组减少了 5 9.8%。

Objective To assess the effect of local transfer of plasmid mediated inducible Nitric Oxide synthase gene using protein coated metallic stents of inhibiting neointimal hyperplasia after coronary angioplasty. Methods The metallic stent was coated by cross linked gelatin and mounted on 3.0 mm PTCA balloon,then endotoxin free ultrapure plasmid pcDNA3hepiNOS was absorbed on the stent. Protein coated stainless steel stents were used as controls. All stents were implanted into the middle segment of LAD. The ratio of balloon to vessel diameter was 1.1-1.3:1. Results At the 7th day after stenting, RT PCR and immunohistochemical staining confirmed the expression of iNOS mRNA and presence of its protein at gene transferred vessels ( n =2), but there was no expression in remote organs. After 3 months of stenting coronary angiograms showed that there was no restenosis in all animals transferred plasmid pcDNA3hepiNOS( n =5) while restenosis developed in all animals of the control group ( n =5). Morphometric analysis showed that lumen diameter loss (0.61±0.30) mm vs (1.58± 0.31 ) mm ( P <0.001), residual lumen diameter (1.00±0.51) mm vs (0.36±0.32) mm ( P <0.05), neointimal area (1.65±0.83) mm 2 vs (2.83±0.83) mm 2 ( P <0.05), mean percentage of area stenosis (26.45±7.45) mm 2 vs (94.2±14.3) mm 2 ( P <0.001) were significantly less than those in control group. The proportion of the neointimal area to media area(I/M) reduced to 59.84% in pcDNA3hepiNOS group. Conclusions Local plasmid mediated human iNOS gene transferred with protein coated metallic stents significantly inhibited intimal hyperplasia,which was usually a causative factor of retenosis after coronary angioplasty, in mini swine model.