基于分子影像技术的光学靶向纳米探针诊断和微波消融治疗三阴性乳腺癌的实验研究

发布时间：2018-05-30 23:22

本文选题：活体荧光成像 + 二氧化硅荧光纳米探针　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：研究背景：分子影像学(Molecular imaging, MI)是近年来随着现代化科学技术和生命医学的飞速发展应用而生的新的学科,是将医学影像技术和分子生物学技术相结合的成像方法。利用多种影像学方法和分子探针,能够在活体状态下对生物过程进行细胞层面和分子水平上,甚至基因水平的定性和定量研究。以研究生物体内生化进程和生理变化为目标的新兴学科。MI最显著特点是：借助处于前沿领域的各种分子探针,利用多种影像学方法,如MRI、CT、核素、PET、超声和光学等影像学技术,选择特异性介质,实现对特定甚至单个细胞、分子的非侵入性显像。目的：本课题组利用自行研发构建的分子靶向探针进行光学成像,以考察其生物相容性及特异靶向性为目的,进行了一系列研究,包括探针的体内体外实验、人三阴性裸鼠乳腺癌模型构建、肿瘤的活体成像诊断及治疗为一体的研究。方法：文章的第一部分采用反相微乳液法制备了介孔二氧化硅(MSNs),以此为载体介质,选用近红外荧光染料吲哚菁绿(ICG)为荧光材料,制备了ICG-MSNs荧光生物化学纳米颗粒,通过共价偶联的方法上载了特异性靶向肿瘤新生血管的靶向配体RGD,获得荧光信号较强的纳米探针RGD-ICG-MSNs 。利用荧光显微镜观察探针对RGD(+)表达的MDA-MB-231三阴性乳腺癌细胞的靶向性,以RGD(-)表达的乳腺癌细胞系MCF-7为对照,考察其用于标记与靶向吞噬性的效能。采用慢病毒转染了荧光素酶和绿色荧光蛋白的人类三阴性乳腺癌MDA-MB-231细胞系,传代培养,接种于BALB/C裸鼠的肩胛部皮下、乳房垫下及尾静静注射,建立三阴性乳腺癌裸鼠皮下、原位及静脉转移性肿瘤模型。文章第二部分利用热层析成像仪和超声仪在肿瘤生长早期进行检测,并用小动物活体成像仪监测肿瘤细胞在裸鼠体内的生长情况及探针的主动靶向性能。在癌细胞接种的第5天时,热层析成像系统报告了该部位的异常表现,而同一组裸鼠在第9天时超声才可以探及结节样结构,因此,热层析成像仪对微小肿瘤的检测要早于超声。荧光靶向探针在小动物活体荧光仪的监视下,不仅可以准确的靶向活体的皮下和原位表浅的乳腺肿瘤,而且可以靶向裸鼠腹腔内的多发转移性淋巴结。文章的第三部分利用所构建的活体肿瘤模型进行超声引导下微波消融治疗,并且术前与术后均用超声造影来评价肿瘤血管的灌注情况及毁损情况,为肿瘤治疗提供了可视化原位灭活的新方法。结果：实验结果表明,RGD(+)表达的MDA-MB-231细胞较RGD(-)表达的乳腺癌MCF-7细胞对荧光纳米颗粒的吞噬效果明显增强;RGD-ICG-MSNs具有生物相容性好、细胞毒性低及化学稳定性好的优点;成功建造了三阴性乳腺癌的裸鼠皮下瘤、原位瘤及静脉转移瘤模型,该动物模型用于后续的热层析成像实验及活体荧光成像实验,用以考察肿瘤细胞的最早检测时间及探针在体内的寻靶性能;热层析成像仪和超声仪在肿瘤生长早期进行检测结果表明,在癌细胞接种的第5天时,热层析成像系统报告了该部位的异常表现,而同一组裸鼠在第9天时超声才可以探及结节样结构,因此,热层析成像仪对微小肿瘤的检测要早于超声;荧光靶向探针可以准确的靶向活体的皮下和原位表浅的乳腺肿瘤,可以靶向裸鼠腹腔内的多发转移性淋巴结;超声引导下微波消融治疗裸鼠皮下及原位肿瘤,并且术前与术后均用超声造影来评价肿瘤血管的灌注情况及毁损情况,力求为肿瘤治疗提供可视化原位灭活的新方法提供实验依据。结论：RGD-ICG-MSNs具有良好的化学稳定性及生物相容性,细胞毒性小,可以用于人三阴性乳腺癌裸鼠皮下及原位肿瘤的靶向诊断;利用热层析及荧光成像系统为肿瘤早期、精准的定性诊断奠定实验基础;超声可视化引导下微波消融可以彻底消灭肿瘤细胞。
[Abstract]:Background: Molecular imaging (MI) is a new discipline developed in recent years with the rapid development of modern science and technology and life medicine. It is an imaging method combining medical imaging technology with molecular biology technology. By using a variety of imaging methods and molecular probes, it can be used in living organisms. The process carries on the qualitative and quantitative study of the cell level and molecular level, and even the gene level. The most prominent feature of the new subject.MI, which aims at studying biochemical processes and physiological changes in the organism, is to use a variety of molecular probes in the frontiers and use a variety of imaging methods, such as MRI, CT, nuclide, PET, ultrasound and optical image. In order to investigate the biocompatibility and specific targeting, a series of studies have been carried out, including the in vitro and in vitro experiments of the probe. Three the construction of the breast cancer model in negative nude mice, the research of the living imaging diagnosis and treatment of the tumor. Method: the first part of the article uses the reverse microemulsion method to prepare the mesoporous silica (MSNs) as the carrier medium, and uses the near infrared fluorescent dye indocyanine green (ICG) as the fluorescent material to prepare the ICG-MSNs fluorescence biochemistry. Nanoparticles, by covalently coupled method, loaded the targeted ligand RGD for specific targeting tumor neovascularization, and obtained a strong nanoscale probe RGD-ICG-MSNs. The targeting of MDA-MB-231 three negative breast cancer cells expressed by RGD (+) was observed by fluorescence microscopy, and MCF-7 of the breast cancer cell line expressed by RGD (-) was the pair. The human three negative breast cancer MDA-MB-231 cell lines, which were transfected with luciferase and green fluorescent protein, were cultured, inoculated in the subcutaneous tissue of the scapula of BALB/C nude mice, under the breast padding and tail injection to build the subcutaneous, in situ and venous transfer of three negative breast cancer in nude mice. The second part of this article is to detect the growth of tumor cells in nude mice and the active target performance of the probe in nude mice by using the thermal tomography instrument and the ultrasound apparatus in the early stage of tumor growth. The thermal layer analysis system reports the abnormal manifestation of the tumor at the fifth day of the cancer cell inoculation. In the same group of nude mice, the nodular structure can be detected by ultrasound at ninth days. Therefore, the detection of microtumors by the thermal tomography instrument should be earlier than that of the ultrasound. Under the monitoring of the small animal living fluorograph, the fluorescent target probe can not only target the subcutaneous and the superficial breast tumors of the living body accurately, but also target the nude mice intraperitoneally. The third part of the article uses the constructed living tumor model for ultrasound guided microwave ablation, and both preoperative and postoperative ultrasound contrast is used to evaluate the perfusion and damage of tumor vessels. A new method of visualizing the in-situ inactivation of tumor is provided for the treatment of tumor. The MDA-MB-231 cells expressed by RGD (+) are more effective than RGD (-) expressed in RGD (-) breast cancer MCF-7 cells, and RGD-ICG-MSNs has the advantages of good biocompatibility, low cytotoxicity and good chemical stability. The subcutaneous tumor of nude mice with three negative breast cancer, in situ tumor and vein metastatic tumor model have been successfully constructed. The object model was used for subsequent thermal tomography and in vivo fluorescence imaging experiments to investigate the earliest detection time of the tumor cells and the searching performance of the probe in the body. The thermal tomography imaging instrument and the ultrasound apparatus were used to detect the early growth of the tumor. The thermal tomography system reported the site at the fifth day of the cancer cell inoculation. The same group of nude mice can detect the nodular structure at ninth days. Therefore, the detection of microtumors by the thermal tomography instrument should be earlier than that of ultrasound; the fluorescent target probe can target the subcutaneous and shallow breast tumor in situ, and can target the multiple metastatic lymph nodes in the abdominal cavity of nude mice; Microwave ablation in the treatment of subcutaneous and orthotopic tumors in nude mice, and the preoperative and postoperative ultrasonography were used to evaluate the perfusion and damage of tumor vessels, and to provide experimental basis for the new method of visualizing in situ inactivation of tumor. Conclusion: RGD-ICG-MSNs has good chemical stability and biocompatibility. Small toxicity, can be used in the target diagnosis of subcutaneous and in situ tumors in nude mice with three negative breast cancer; using thermal tomography and fluorescence imaging system for early tumor, accurate qualitative diagnosis lay the experimental basis; ultrasound visualization guided microwave ablation can completely eliminate tumor cells.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9;R445

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