论文部分内容阅读
目的通过化学和免疫学双重偶联,构建支架结合基因的血管内基因转运体系,评价其可行性及效果。方法采用双官能偶联剂N-琥珀酰亚胺基3-(2-吡啶二硫基)丙酸酯将特异性抗腺病毒六位体(Fab)2’片段以化学键结合在胶原涂层的支架上,编码绿色荧光蛋白的腺病毒作为报告基因载体,通过免疫作用连接在结合了(Fab)2’的支架上,分别在体外和体内实验中验证其效果。结果体外实验结果发现,实验组支架胶原涂层的表面有大量编码绿色荧光蛋白的腺病毒转染的细胞浸润生长,与支架接触的培养皿表面生长的细胞转染率高达92.8%±2.5%,而未与支架直接接触的周边细胞几乎没有被转染。转染率与编码绿色荧光蛋白的腺病毒的反应用量在107~1010病毒粒子范围内呈直线关系,有明显的量效对应性。猪冠状动脉支架植入实验中,回收的支架上和与支架接触的血管组织内有广泛的绿色荧光蛋白表达,血管组织中总的转染率占细胞总数的5.9%±1.1%,转染细胞主要集中在新生内膜(>17%)和中膜(>7%),外膜几乎没有转染。远隔器官(肺、肝、肾)和下游冠状动脉样本未见绿色荧光蛋白DNA表达。结论通过化学偶联的方法在支架上固定抗腺病毒特异性抗体结合腺病毒的基因转运体系可局部靶向、高效地进行血管内介入性基因转运。
OBJECTIVE: To construct intravascular gene delivery system of scaffold-binding genes by double-coupling of chemistry and immunology, and to evaluate its feasibility and effectiveness. Methods The specific anti-adenovirus hexa (Fab) 2 ’fragment was chemically bonded to the collagen coated layer using the bifunctional coupling agent N-succinimidyl 3- (2-pyridyldithio) propionate On the scaffold, the adenovirus encoding green fluorescent protein (EGFP) was used as a reporter vector and was linked by immunization to (Fab) 2 ’scaffolds to verify its efficacy in vitro and in vivo, respectively. Results The results of in vitro experiments showed that a large number of cells transfected with adenovirus encoding green fluorescent protein (GFP) were infiltrated on the surface of the experimental collagen coating. The transfection rate of the cells growing on the surface of the petri dish contacted with the stent was as high as 92.8% ± 2.5% Peripheral cells that were not in direct contact with the scaffold were almost not transfected. The transfection rate was linear with the amount of adenovirus encoding green fluorescent protein in the range of 107-1010 virus particles with significant dose-response. In the pig coronary artery stent implantation experiment, a wide range of green fluorescent protein expression was found in the recovered scaffolds and the vascular tissues contacted with the scaffolds. The total transfection rate in the blood vessel tissues accounted for 5.9% ± 1.1% of the total number of the cells. The transfected cells Mainly in the neointima (> 17%) and the media (> 7%), the outer membrane almost no transfection. GFP expression was not observed in distant organs (lung, liver, kidney) and downstream coronary arteries. Conclusion The method of chemical conjugation immobilizes the anti-adenovirus-specific antibody combined with the adenovirus gene delivery system on the scaffold to locally and efficiently target the intravascular gene delivery.