涂覆承载质粒DNA水凝胶的覆膜血管内支架置入主动脉局部基因转染的实验研究

目的 用部分覆膜血管内支架涂覆阳离子胶原水凝胶(CGH)承载质粒DNA实现主动脉局部的基因转染,为核酸等大分子治疗物质经血管导入提供理论依据.方法 将CGH涂覆于部分覆膜血管内支架的覆膜织物上,以CGH涂层分别承载2种质粒DNA,即编码β-半乳糖苷酶的pCAGGS-LacZ或编码增强绿色荧光蛋白的pEGFP.8只实验兔为日本白色家兔,在主动脉经球囊拖拉损伤4周后置入2枚血管内覆膜支架,分别承载pCAGGS-LacZ和pEGFP.因2枚支架承载的质粒DNA不同,转染后转录的mRNA和表达的蛋白不同,所以2枚血管内覆膜支架置入部位主动脉互为阴性对照.术后3 d对基因转染和表达进行评估.pCAGGS-LacZ的转染和表达通过特异性x-gal染色和反转录聚合酶链反应(RT-PCR)检测.pEGFP的转染和表达通过免疫荧光显微镜观察.结果 在承载pCAGGS桳acZ的CGH涂层部分覆膜血管内支架置入部位的主动脉,尤其是金属支架支杆对覆膜材料支撑良好处主动脉局部x梘al染色阳性,RT桺CR可检测到编码β-半乳糖苷酶的mRNA;免疫荧光显微镜观察未见绿色荧光,该处主动脉近端及远端x-gal染色及RT-PCR结果均为阴性.其他器官,如脑、心、肝、RT.PCR结果均为阴性.在承载pEGFP的CGH涂层部分覆膜血管内支架置入部位主动脉局部,免疫荧光显微镜可观察到绿色荧光;x梘al染色阴性,RT-PCR未检测到编码β-半乳糖苷酶的mRNA.结论 CGH涂层部分覆膜血管内支架置入兔主动脉内,可实现主动脉局部的瞬时基因转染与表达.

Gene delivery of plasmid DNA to rabbit aorta by genetic engineering of cationized gelatin hydrogel coated partially covered endovascular stent graft

Objective To genetically engineer endovascular stent grafts that facilitate plasmid DNA delivery and offer the promise ofcular delivery system of therapeutic materials. Methods Partially covered polyester stent-grafts coated with cationized gelatin hydrogels (CGH) containing pCAGGS-LacZ or pEGFP were implanted in the descending aorta of 8 rabbits, which had neointima due to balloon injury four weeks ago. The aorta with stent-graft containing pCAGGS-LacZ and the one containing pEGFP was taken as negative control for each other. Expression of the plasmid-encoded marker genes, (3-galactosidase and enhanced green fluorescence protein (EGFP) were evaluated at 3 days after implantations by X-Gal staining and RT-PCR or fluorescence microscopy. Results Local plasmid DNA transfer was confined to the vessel wall at the site of stent-graft implantation, especially where the graft was compressed firmly to the vessel by metal struts. Plasmid DNA was not detected in vessel segments immediately proximal or distal to the stent graft and dissemination of plasmid DNA to brain, heart, lung, liver or kidney was not observed. The (3-galactosidase-expressed cells were identified as endothelial cells, and smooth muscle cells by pathological analysis. Fluorescence microscopy identified the EGFP expression which demonstrated the transgene delivery of plasmid DNA and it was not related to the plasmid-encoded marker genes. No signal was detected in the aorta of the rabbits that received cationized gelatin hydrogels coated stent-grafts without plasmid DNA. Conclusions Cationized gelatin hydrogels coated partially covered stent grafts provide a new access for transgene delivery to the cells of aortic wall.