血管生成靶向微泡的鉴定及黏附实验

Identification and adhesion experiment of microbubbles targeted to angiogenesis

目的 体外评价＂亲和素-生物素＂法制备靶向整合素αvβ3的微泡（MBαvβ3）及其黏附人脐静脉内皮细胞（HUVECs）的效能.方法 ＂亲和素-生物素＂法桥连αvβ3抗体于磷脂微泡,体外荧光法鉴定其表面＂生物素-亲和素-生物素＂结构,应用平行平板流动腔（PPFC）技术评价MBαvβ3特异性黏附HUVECs的效能.结果 加入荧光标记的亲和素后,生物素化微泡（MBB）表面可见明亮荧光,普通微泡未见荧光;MBB加入荧光标记的蛋白A也未见荧光;MBB结合亲和素后,再加入荧光标记的生物素或蛋白A,前者表面可见明亮荧光,后者未见荧光.PPFC内,MBαvβ3组和普通微泡组结合数分别为（9.9±3.1）微泡/HUVEC和（0.8±0.3）微泡/HUVEC（P〈0.05）.结论 ＂生物素-亲和素＂法能成功制备MBαvβ3,具有特异黏附血管内皮细胞的能力.

Objective To identify microbubbles targeted （MBt） to alpha（v）beta（3） （αvβ3） via biotin-avidin bridge and evaluate the adhesion to human umbilical vein endothelial cells （HUVECs） in vitro.Methods MBt produced via biotin-avidin bridge were validated using fluorescence in vitro.Adhesion of αvβ3-integrin targeted MBt （MBαvβ3） to HUVECs was tested using the parallel plate flow chamber （PPFC） test.Results Bright green fluorescence was observed on the biotinylated microbubbles（MBB） incubated with fluorescein isothiocyanate labeled streptavidin （FITC-SA） and on MBB-SA incubated with FITC labeled biotin.There was no fluorescence seen on non-targeted control microbubbles,MBB incubated with FITC labeled protein A and MBB-SA incubated with FITC labeled protein A. The adherent rate of MBαvβ3 was significantly higher than MBt with non-specific antibody （MBN） in PPFC test,with 9.9±3.1 of MBαvβ3 and 0.8±0.3 of MBN adhered to HUVECs,respectively（P〈0.05）.Conclusions Avβ3 targeted microbubbles using biotin-avidin bridging method is highly efficient and reliable for HUVECs.