摘要
目的本研究拟制备携载G250单克隆抗体的纳米微泡,在体内和体外研究其对肾细胞癌的靶向性。方法机械振荡法制备空白脂质纳米微泡并观察其稳定性,生物素-亲和素连接技术将纳米微泡与G250单克隆抗体相连,采用免疫荧光进行验证。细胞免疫荧光实验鉴定所使用的两种肾细胞癌细胞(786-O和ACHN细胞)中G250的表达情况,靶向结合实验鉴别靶向纳米微泡对786-O和ACHN两种肾细胞癌细胞的体外寻靶能力。在肾细胞癌的裸鼠皮下移植瘤模型中,使用纳米微泡进行超声造影,并将采集到的动态图像在定量分析软件Qlab 8.1中进行数据分析。结果成功制备了平均粒径为404.9 nm空白纳米微泡和平均粒径为611.4 nm靶向纳米微泡,稳定性能好,同时免疫荧光证实了靶向纳米微泡构建成功,能够与FITC标记的二抗结合,从而显示出绿色荧光;细胞免疫荧光证实G250抗原在786-O细胞中呈膜表达,而ACHN细胞中不表达。在细胞结合实验中发现靶向纳米微泡能够特异性的结合786-O细胞,但不能结合于ACHN细胞。体内超声造影显像从平均值和最大值分析,靶向纳米微泡和空白微泡在786-O肾细胞癌细胞的裸鼠模型的显影效果存在显著差异[平均值:(11.74±0.52)vs(16.34±0.40),P=0.001;最大值:(13.07±0.94)vs(18.09±0.82),P=0.003]。结论 G250靶向的纳米微泡可在体外能够特异性的结合G250表达阳性的肾细胞癌细胞,在动物体内能够明显的增强移植瘤的超声显影效果。
Objective To prepare targeted nanoscale microbubbles（ NBs） with G250 monoclonal antibodies targeting renal cell carcinoma（ RCC）,and explore its targeting ability to RCC in vitro and in vivo.Methods Mechanical oscillation method was used for preparing blank NBs and its stability was observed.Targeted NBs was prepared by biotin-avidin linking technology and verified by immunofluorescence assay. The expression of G250 in 2 kinds of RCC cells（ 786-O and ACHN） was identified by immunofluorescence assay.Targeting ability of the 2 kinds of NBs in vitro was identified by target binding assay. In the nude mice model of RCC,contrast-enhanced ultrasound imaging was used for imaging the 2 kinds of NBs,and the dynamic images were collected and analyzed in the quantitative analysis software Qlab 8. 1. Results The blank NBs and targeted NBs in average particle sizes of 404. 9 nm and 611. 4 nm,respectively,were successfully prepared,and with good stability. The targeted NBs were labeled with FITC, thus showing green fluorescence. Immunofluorescence assay confirmed that membrane antigen G250 was expressed in 786-O cells,but not expressed in ACHN cells. In the cell binding experiment,it was found that the targeted NBs were able to specifically bind to 786-O cells,but unable to bind to ACHN cells. In vivo ultrasound contrast imaging showed that the mean value and maximum value of the blank NBs and targeted NBs in 786-O cells were significantly different from those in ACHN cells in the nude mice model（ average mean： 11. 74 ± 0. 52 vs16. 34 ± 0. 40,P = 0. 001; maximum value： 13. 07 ± 0. 94 vs 18. 09 ± 0. 82,P = 0. 003）. Conclusion The prepared targeted NBs targeting G250 can specially bind to 786-O cells in vitro, and significantly enhance the ultrasound imaging effect in vivo.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第2期112-118,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81472399)~~
关键词
纳米微泡
超声增强显影技术
肾细胞癌
单克隆抗体
nanoscale microbubbles
G250
contrast-enhanced ultrasound imaging
renal cell carcinoma
monoclonal antibodies
