载STEAP-1及VEGFR-2抗体前列腺癌靶向超声微泡的制备及寻靶实验研究

发布时间：2018-03-12 18:29

本文选题：前列腺跨膜上皮1抗原　切入点：血管内皮生长因子受体　出处：《皖南医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:使用前列腺跨膜上皮抗原-1(STEAP-1)抗体及血管内皮生长因子受体2(VEGFR-2)抗体作为配体,采用生物素-亲和素法制备携带上述抗体的新型靶向超声微泡造影剂,并探讨其体外寻靶能力,分析所制备造影剂在荷人前列腺癌裸鼠增强显影效果,希望获得有价值的早期超声诊断方法。方法:(1)采用生物素-亲和素方法制备靶向超声造影剂,将STEAP-1抗体及VEGFR-2抗体与普通超声造影剂进行结合,并在荧光显微镜下观察微泡荧光情况,并通过流式细胞仪检测其结合率及稳定性。(2)在无菌环境下体外培养人前列腺癌细胞株PC3并传代,在雄性裸鼠皮下接种PC3细胞,建立荷人前列腺癌移植瘤雄性裸鼠模型。通过荷瘤裸鼠模型尾静脉注射靶向超声微泡造影剂及普通超声造影剂进行超声增强显影,观察裸鼠移植瘤的造影剂分布特征及实时灌注情况,并用相关软件绘制时间-强度曲线进行定量分析,比较不同造影剂的达峰时间(Time to Peak,TTP)、峰值强度(Peak Intensity,PI)及平均渡越时间(Mean Transit Time,MTT)。结果:(1)携带STEAP-1抗体及VEGFR-2抗体的靶向微泡超声造影剂通过免疫荧光实验在荧光显微镜下均呈阳性表达,即微泡边缘出现绿色荧光。(2)成功建立荷人前列腺癌裸鼠模型,通过鼠尾静脉注射普通超声造影剂及两种靶向造影剂,所有组别均可见明显增强,达到一定峰值后逐渐减弱。结论:(1)采用生物素-亲和素法可以成功制备携带STEAP-1抗体及VEGFR-2抗体的靶向超声造影剂,且两种靶向超声造影剂性质均较稳定;(2)在体外实验中,携带STEAP-1抗体及VEGFR-2抗体的靶向超声造影剂在荷瘤裸鼠中靶向性较好且特异性较高,其中VEGFR-2抗体可以与前列腺癌新生血管内皮细胞特异性结合,为评估前列腺癌新生血管形成提供了新的思路。
[Abstract]:Objective: to prepare a new type of ultrasound microbubble contrast agent carrying the antibodies against prostate transmembrane epithelial antigen-1 (STEAP-1) and vascular endothelial growth factor receptor 2VEGFR-2 (VEGFR-2) by biotin-avidin method. The aim of this study was to investigate the ability of target finding in vitro, to analyze the enhancement effect of the contrast agent in nude mice bearing human prostate cancer, and to obtain a valuable method for early ultrasound diagnosis. Methods: biotin-affinity method was used to prepare the target ultrasound contrast agent. STEAP-1 antibody and VEGFR-2 antibody were combined with conventional ultrasound contrast medium, and the fluorescence of microbubbles was observed under fluorescence microscope. The binding rate and stability of human prostate cancer cell line PC3 were detected by flow cytometry. PC3 cells were cultured and subcultured in vitro. PC3 cells were subcutaneously inoculated into male nude mice. The male nude mice model of transplanted tumor of human prostate cancer was established. Ultrasound enhanced imaging was performed by injection of targeted ultrasound microbubble contrast agent and conventional ultrasound contrast agent into tail vein of nude mice bearing human prostate cancer. The distribution characteristics of contrast media and the real-time perfusion of transplanted tumor in nude mice were observed and the time-intensity curves were plotted by relevant software for quantitative analysis. The peak time and peak intensity of different contrast agents were compared. The peak intensity and the mean transit time were compared. The results showed that the microbubble ultrasound contrast agents carrying STEAP-1 antibody and VEGFR-2 antibody were all positive under fluorescence microscope. That is, green fluorescence appeared at the edge of the microbubble. (2) A nude mouse model of human prostate cancer was successfully established. The normal ultrasound contrast agent and two target contrast agents were injected into the tail vein of the mouse, and the enhancement was observed in all groups. Conclusion: in vitro, STEAP-1 antibody and VEGFR-2 antibody targeting ultrasound contrast agent can be successfully prepared by using biotin-avidin method, and the properties of both targeted ultrasound contrast agents are stable in vitro. The ultrasound contrast agent carrying STEAP-1 antibody and VEGFR-2 antibody was more specific and specific in tumor-bearing nude mice. VEGFR-2 antibody could specifically bind to prostate cancer neovascularization endothelial cells. It provides a new idea for evaluating the angiogenesis of prostate cancer.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R445.1;R737.25

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