金属蛋白涂层支架用于小型猪冠状动脉质粒介导下转基因研究

Plasmid-mediated Intracoronary Local Gene Transfer Using Protein coated Metallic Stent in Mini swine

目的 :评价在质粒介导下 ,金属蛋白涂层支架向血管内局部转基因的可行性、效率和选择性。 方法 :金属支架由 316 L不锈钢丝编织而成 ,其涂层是通过把支架放入有交联剂的明胶溶液中浸泡而成。基因载体为Pc DNA2质粒 ,并携带有β-半乳糖苷酶标记基因 ,该基因编码核特异性β-半乳糖苷酶。首先将蛋白涂层支架分别固定在3.0 mm或 3.5 mm经皮冠状动脉腔内成形术球囊上并在浓度为 8μg/μl的基因原液中浸泡 3分钟 ,然后通过 8F大腔引导导管将支架送入小型猪冠状动脉前降支中段 (转基因组 ,n=3) ,另外把没有浸泡过基因的支架也送入小型猪冠状动脉前降支中段 (对照组 ,n=3)。在支架植入后 7天处死动物。β-半乳糖苷酶表达由 X- Gal染色评估。 结果 :所有转基因动物均有基因表达。转基因表达出现在内膜、中层和外膜。中层平滑肌细胞转染率为 3.0 %。远处器官和对照组冠状动脉均未显示核特异性β-半乳糖苷酶阳性表达。 结论 :蛋白涂层支架在质粒介导下向血管内转基因有效、可行 ,因此 ,它有可能成为冠状动脉腔内成形术后再狭窄基因治疗的有效转基因系统。

Objective:To assess the feasibility,efficiency and selectivity of plasmid mediated gene transfer to local arterial wall by protein coated metallic stent. Method:Metallic stent was made by 316L stainless wire.The coating for metallic stent was made by immersing in gelatin solution containing crosslinker.PcDNA2 plasmid carrying LacZ reporter gene for nuclear specific β galactosidase was used.The coated stent was mounted on a 3 5 or 3 0 mm percutaneous transluminal coronary angioplasty (PTCA) balloon and immersed into the gene stock(8 μg/μl)for 3 min,and then implanted into the left anterior descending branch of coronary artery of mini swine( n =3)via a 8F large lumen guiding catheter.Coated stent without gene was implanted into the same of mini swine as control( n =3).All the animals were sacrificed 7 days after implantation.β galactosidase expressioon was assessed by X gal staining. Results:The expression of transgene was detected in the intima,media and adventitia of all the transgenic animals.The transfection efficiency of medial smooth muscle cells was 3 0%.No X gal stained samples was detected in remote organs and coronary arteries of the controls. Conclusions:Plasmid mediated arterial gene transfer to endothelial,smooth muscle cells and adventitia is feasible and efficacious by protein coated metallic stent.The coated stent may be a good carrier for plasmid mediated gene transfer and thus a potentially effective transgenic means for the prevention of restenosis following PTCA.