载肝癌细胞GPC3抗体的纳米级脂质超声微泡制备及体外寻靶研究  

Preparation of nanoscale lipid microbubbles modified with hepatoma glypican 3 antibody and targeting study in vitro

作　　者：景周宏[1] 何承俊[1] 潘万龙[2] 丁雄[1] 刘长安[1] 李生伟[1] 薛强[1] 龚建平[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,重庆400010 [2]重庆医科大学感染病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2013年第5期449-453,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金面上资助项目(编号:81272570);重庆市卫生局重点资助项目(编号:渝卫2012-1-040)

摘　　要：目的：探索制备载肝癌细胞磷酯酰肌醇蛋白多糖-3（glypican-3,GPC3）抗体的新型纳米级脂质超声微泡造影剂的方法,并评价其体外对肝癌细胞的靶向结合效果。方法：以机械振荡法制备纳米级脂质微泡,并通过光学显微镜及电子显微镜检测纳米微泡大小形态、粒径分布;生物素-亲和素桥接肝癌细胞GPC3抗体与纳米级脂质超声微泡,流式细胞仪检测微泡与抗体结合效率;免疫组化检测常见3种肝癌细胞的GPC3阳性表达;微泡体外与肝癌细胞靶向结合,激光共聚焦显微镜检测寻靶效果。结果：纳米脂质微泡呈圆形,分布均匀,无明显聚集,粒径范围约350~450 nm。微泡与抗体结合效率达85.05%,人SMMC-7721、HepG2及Huh7肝癌细胞GPC3表达阳性,激光共聚焦显微镜显示载GPC3抗体纳米级脂质微泡可与肝癌细胞特异性结合。结论：本研究成功制备了具备靶向性的新型纳米级脂质超声微泡,生物素-亲和素桥接GPC3抗体可特异性结合肝癌细胞,达到靶向性识别效果,为微泡的靶向诊断及治疗研究奠定了基础。Objectives：To develop a method for the preparation of hepatoma glypican 3 （GPC3） antibody modified lipid microbubble and to evaluate its efficiency of targeting hepatoma cells in vitro. Methods ： Lipid microbubbles were prepared using the mechanical oscil- lation method and the morphology and size of the microbubbles were determined using optical and electron microscopy. The biotin-a- vidin bridge was used to link the GPC3 antibody with the microbubbles and the efficiency of conjugation was determined using flow cytometry. Expressions of GPC3 in 3 kinds of common hepatoma cells were investigated using the immunocytochemical assay. GPC3 antibody modified microbubbles were targeted to hepatoma cells in vitro and the targeting efficiency was determined using confocal analysis. Results：Lipid microbubbles showed a round shape and were evenly distributed without significant aggregation. Diameter of the microbubbles was between 350 nm and 450 nm. 85.05% microbubbles were successfully conjugated with GPC3 antibodies. Fur- thermore,SMMC-7721, HepG2 and Huh7 cells showed positive GPC3 expressions and confocal analysis revealed that the microbub- bles could specifically bind to SMMC-7721 cells. Conclusions：Nanoscale lipid microbubbles conjugated with GPC3 antibody are successfully obtained via biotin-avidin bridging. GPC3 antibody modified microbubbles could selectively target hepatoma cells. Our study provides experimental support for the application of antibody modified nanoscale microbubbles in diagnosis and treatment.

关 键 词：磷酯酰肌醇蛋白多糖-3抗体 纳米级 超声微泡 肝癌细胞 体外寻靶 

分 类 号：R735.7[医药卫生—肿瘤] R445.1[医药卫生—临床医学]