穿膜肽与P-选择素抗体修饰的载基因超声造影剂的制备及体外靶向实验研究  

Preparation and Targeting Study of Gene-loaded Ultrasound Contrast Agents Modified with Cell-penetrating Peptides and P-selectin Antibody

作　　者：田菊[1] 王志刚[1] 任建丽[1] 王星[1] 

机构地区：[1]重庆医科大学超声影像学研究所,重庆市400010

出　　处：《中国超声医学杂志》2012年第6期481-484,共4页Chinese Journal of Ultrasound in Medicine

基　　金：国家自然科学基金青年科学基金项目(No.81000621);国家自然科学基金面上项目(No.81071158);国家自然科学基金重点项目(No.81130025);国家重点基础研究发展计划(973计划)(No.2011CB707900)

摘　　要：目的制备穿膜肽与P-选择素抗体修饰的载基因超声造影剂,并在体外评价其靶向结合能力。方法采用机械振荡法制备载基因及穿膜肽超声造影剂,激光共聚焦显微镜观察质粒DNA和穿膜肽的分布,并用碳二亚胺法制备P-选择素靶向超声造影剂,通过免疫荧光法检测其与抗体的连接情况。制备人脐静脉内皮细胞(HUVEC)缺氧模型,光镜及流式细胞仪检测该靶向超声造影剂与缺氧HUVEC的结合情况。结果激光共聚焦显微镜可以观察到穿膜肽和质粒DNA均匀地分布在超声造影剂壁上。通过免疫荧光法在荧光显微镜下观察到靶向超声造影剂表面呈红色荧光。体外寻靶实验显示,光镜下靶向组超声造影剂牢固地结合在缺氧HUVEC周围,而非靶向组未见超声造影剂结合;流式细胞仪检测靶向组大约有73.80%的细胞表面结合有该靶向超声造影剂。结论成功制备了穿膜肽与P一选择素抗体修饰的载基因超声造影剂,并具有较强的靶向结合能力。Objective To prepare a kind of gene-loaded ultrasound contrast agents （UCA） modified with cell- penetrating peptides （CPP） and P-selectin antibody and to evaluate their targeting affinity for hypoxia human umbilical vein endothelial cell（HUVEC） in vitro. Methods The UCA carrying plasmid DNA and CPP was prepared using me- chanical vibration. The distribution of plasmid DNA and CPP on the UCA was investigated using confocal laser scan- ning microscopy（CLSM）. Anti-P-selectin antibody was covalently linked to the surface of UCA using a carbodiimide technique. The connection of anti-P-selectin antibody with UCA was detected by immunofluorescent assay, and targe- ting specifity of the UCA to hypoxia HUVEC was observed by light microscope and flow cytometry （FCM）. Results The results of CLSM showed that plasmid DNA and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using fluorescent microscopy. The conjugation of the targeted UCA with hy- poxia HUVEC was firm, while the conjugation in control group was negative. The result of FCM showed that about 73.80~//oo of the cell surface combined with the targeted UCA. Conclusions The gene-loaded UCA modified with CPP and P-selectin antibody was prepared successfully. The targeted UCA had a strong target binding capacity to hypoxia HUVEC.

关 键 词：穿膜肽 基因转染 超声造影剂 靶向 

分 类 号：R445.1[医药卫生—影像医学与核医学]