冠脉搭桥手术中桥血管模型建立及内皮型一氧化氮合酶基因转染的研究

The establishment of bridge conduits model for artery grafts in coronary artery bypass grafting and experimental study for adenoviral-mediated gene transfection of endothelial nitric oxide synthase

目的建立冠状搭桥手术（CABG）中桥血管动物模型，桥血管壁转染人内皮型一氧化氮合酶（eNOS）基因并验证转染效果。方法家兔40只，随机分为4组（n=10），自体股动脉移植建立动脉桥血管模型：（1）eNOS组，股动脉转染eNOS基因后移植为桥血管；（2）空载基因组，股动脉转染空载eNOS基因后移植为桥血管；（3）对照组，股动脉仅移植为桥血管，不进行基因转染；（4）正常股动脉组。基因转染采用浸泡法转染腺病毒封装的eNOS基因组或空载eNOS基因组。3周后获取桥血管、正常颈动脉以及股动脉标本，通过免疫组织化学法检测组织中一氧化氮（NO）含量、eNOS活力测定和eNOS蛋白采用Western blot等方法，观察和证实转染的效果和效率。结果本组兔无死亡，桥血管通畅。模型中各组桥血管均可见不同程度的适应性改变。其中eNOS组血管壁增厚，eNOS蛋白表达明显增多；对照组和空载基因组可见血管壁变性，eNOS蛋白不表达或表达极少（P〈0．05）。eNOS组动脉管壁NO含量及总eNOS活力分别为（8．83±1．24）μmol／g和（0．987±0．239）U／mg，远高于其他3组（P〈0．05）。Western blot分析也证明eNOS组血管壁eNOS蛋白含量明显增多，与其他3组比较差异有统计学意义（P〈0．05）。结论此动物模型有一定实用性。可以借助eNOS基因转染增加动脉桥血管壁eNOS表达及活力，进而增加NO在局部的含量及表达。

Objective The experiment was designed .to establish the animal model of bridge conduits for artery grafts in coronary artery bypass grafting （CABG） and evaluate the efficacy of adenoviral-mediated gene transfection with endothelial nitric oxide synthase （eNOS）. Methods Forty rabbits were divided into 4 groups （n = 10） randomly. Establish the bridge conduits model by end-to-end anastomosis of the femoral artery to carotid artery ： （ 1 ） eNOS group, the bridge graft was transferred with adenoviral-mediated eNOS; （2） zero load group, adenoviral-mediated zero load eNOS was transferred; （3） control group, only the bridge graft without gene transfection; （4） normal femoral artery group. Gene transfection was by the way of soaking method for adenoviral-mediated eNOS gene or zero load eNOS gene for 1 h. Three weeks later, the grafts were harvested and sent for measurement such as immunohistochemisty, NO content, efficacy of eNOS in the vessel wall and immunoblotting assay （Western blotting）. Results There were no deaths in the rabbits. All of the bridge vessels in different groups got adaptive changes without obstruction. In the eNOS group, the smooth muscle were found thicked and the expression of eNOS increased obviously, while the vascular wall degeneration could be found in the other groups. Also there were no obvious expression of eNOS in the vessel walls in the three groups （P 〈 0. 05 ）. The NO content and eNOS energy in eNOS group were （ 8.83 ± 1.24） μmol/g and （0. 987 ±0. 239） U/mg, different remarkably with other groups （ P 〈 0. 05 ）. The Western blot also show the same results. Conclusion The model we built is reliable and the adenoviral-mediated of eNOS gene transfection is successful, which can be used to increase the content of NO or the express efficacy of eNOS gene in the vessel wall of the artery bridge.