论文部分内容阅读
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: This study aimed to prepare nanometer microbubbles carrying G250 monoclonal antibody to study its targeting to renal cell carcinoma in vitro and in vivo. Methods Blank lipid nano-microbubbles were prepared by mechanical shaking method and their stability was observed. The biotin-avidin connection technique was used to connect nano-microvesicles with monoclonal antibody G250 and validated by immunofluorescence. Cell immunofluorescence assay identified the expression of G250 in two types of renal cell carcinoma cells (786-O and ACHN cells) that were used. Targeted binding experiments were performed to identify the effect of targeted nano-microbubbles on 786-O and ACHN renal cell carcinoma cells In vitro targeting ability. In renal cell carcinoma model of subcutaneous xenograft in nude mice, nanoemulsion was used to make contrast-enhanced ultrasound and the dynamic images were analyzed by quantitative analysis software Qlab 8.1. Results The nanofibers with the average diameter of 404.9 nm and the targeted nanofibers with an average diameter of 611.4 nm were successfully prepared, and the stability was good. Meanwhile, the immunofluorescence confirmed the successful construction of the targeted nanofibers, Anti-binding, which showed green fluorescence; cell immunofluorescence showed that G250 antigen was expressed in 786-O cells membrane, but not in ACHN cells. In cell-binding experiments, it was found that targeted nanobubbles could specifically bind to 786-O cells but not to ACHN cells. In vivo echocardiographic imaging From the mean and maximum values, there was a significant difference in the developmental effects of targeted nanobubbles and blank microbubbles in nude mice bearing 786-O renal cell carcinoma cells [mean: (11.74 ± 0.52) vs (16.34 ± 0.40), P = 0.001; maximum: (13.07 ± 0.94) vs (18.09 ± 0.82), P = 0.003]. Conclusion G250-targeted nano-microbubbles can specifically bind G250 positive cell lines in vitro and can significantly enhance the ultrasound imaging of xenografts in animals.