氧化铁共修饰的明胶硅氧烷纳米粒载体的构建及其基因转染的实验研究
发布时间：2018-09-01 20:04
【摘要】：脑胶质瘤是神经外科中最常见的颅内肿瘤,发病率约占中枢神经系统肿瘤的40%,成人神经恶性肿瘤的78%[1]。近30年来,虽然神经影像学及胶质瘤的治疗都取得了很大进展,但恶性胶质瘤的预后仍然悲观。成人中恶性胶质瘤的1年及5年生存率分别约为30%和13%,其中GBM的中位生存时间仅12月左右[2]。而肿瘤的发生从根本上讲是基因的病变,目前基因治疗从理论到实践为彻底地攻克肿瘤,改善患者预后带来了极大的希望。但仍然有很多环节亟待研究和早日攻破,其中由于缺乏高效稳定的载体系统,加之中枢神经系统血脑屏障结构的特殊性,是导致其治疗效率不高的重要原因之一。 本实验中,通过两步溶胶-凝胶法,初步合成了明胶-硅氧烷纳米粒子(GS NPs),并在此基础上构建了一种经亲水性聚乙二醇(PEG)、阳离子多肽Tat、核酸适配体TTAl和Fe304共修饰的纳米粒子基因载体系统。它不仅能透过血脑屏障,特异靶向脑部肿瘤细胞,并具有MRI显影效果。考察其携带质粒DNA在细胞水平的转染效率,为该纳米粒子作为入脑转运载体的应用提供理论和实践支持。 第一部分通过傅里叶变换红外光谱(FT-IR)表征GS NPs与Fe304的偶联。通过对纳米颗粒的表面电位分析(Zeta potential)、透射电子显微镜(TEM)、纳米颗粒粒径分析(DLS)、傅里叶变换红外光谱(FT-IR)、热失重分析(TGA)等理化分析检测方法,研究该材料的理化特性。 第二部分通过激光共聚焦、MRI等成像技术,验证GS NP-Fe3O4作为基因载体可被C6细胞有效摄取,并能携带GFP (green fluorescent protein)报告基因在肿瘤细胞中高效靶向表达。使用磁共振成像对经过细胞共培养后一定浓度范围内的GS NP-Fe3O4和Fe304进行T2加权序列扫描并分别测量其弛豫率r2值,验证GS NP-Fe3O4作为MRI对比剂的可能性。 第三部分通过在脑立体定位仪引导下注射C6胶质瘤细胞的方法建立大鼠脑胶质瘤模型,之后经尾静脉注射分别GS NPs-Fe3O4和Fe304,比较不同纳米粒子之间的MRI成像效果,观察信号变化情况。对肿瘤标本切片进行HE和普鲁士蓝染色,检测其病理学改变。通过对大鼠C6脑胶质瘤切片及MRI成像检测,探讨GS NPs-Fe3O4是否具有特异性靶向标记成像的特性,进而能够在肿瘤诊断、药物转运及靶向治疗等方面发挥巨大优势。
[Abstract]:Glioma is the most common intracranial tumor in neurosurgery, accounting for 40% of central nervous system tumors and 78% of adult neuromalignant tumors. In recent 30 years, although neuroimaging and glioma treatment have made great progress, the prognosis of malignant gliomas is still pessimistic. The 1-year and 5-year survival rates of adult malignant gliomas were about 30% and 13%, respectively. The median survival time of GBM was only about 12 months [2]. At present, gene therapy from theory to practice has brought great hope to thoroughly conquer the tumor and improve the prognosis of patients. However, there are still many links to be studied and broken down as soon as possible, among which the lack of efficient and stable carrier system and the particularity of blood-brain barrier structure in central nervous system are one of the important reasons leading to the inefficiency of its treatment. In this experiment, a two-step sol-gel method was used. The gelatin-siloxane nanoparticles (GS NPs),) were synthesized and a novel carrier system was constructed on the basis of which an aptamer TTAl and Fe304 were co-modified by polyethylene glycol (PEG), cationic polypeptide Tat, aptamer. It can not only penetrate the blood-brain barrier and specifically target brain tumor cells, but also have the effect of MRI imaging. The transfection efficiency of the plasmid DNA was investigated at the cell level to provide theoretical and practical support for the application of the nanoparticles as brain transporter. In the first part, the coupling of GS NPs and Fe304 was characterized by Fourier transform infrared spectroscopy (FT-IR). The physicochemical properties of the nanoparticles were studied by means of surface potential analysis, (Zeta potential), transmission electron microscope, (TEM), particle size analysis, (DLS), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). In the second part, laser confocal MRI and other imaging techniques were used to verify that GS NP-Fe3O4 as a gene vector could be effectively ingested by C6 cells, and GFP (green fluorescent protein) reporter gene could be efficiently expressed in tumor cells. Magnetic resonance imaging (MRI) was used to scan GS NP-Fe3O4 and Fe304 in a certain concentration range after cell coculture and the relaxation rate R2 was measured respectively to verify the possibility of GS NP-Fe3O4 as a contrast agent for MRI. In the third part, C6 glioma model was established by injecting C6 glioma cells under the guidance of stereotactic locator, and then the MRI imaging effects between different nanoparticles were compared by injecting GS NPs-Fe3O4 and Fe304, respectively through tail vein. Observe the change of signal. HE and Prussian blue staining were used to detect the pathological changes. By detecting rat C6 glioma slices and MRI imaging, this paper discusses whether GS NPs-Fe3O4 has the characteristics of specific targeted imaging, and can play a great advantage in tumor diagnosis, drug transport and targeted therapy.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2218204
[Abstract]:Glioma is the most common intracranial tumor in neurosurgery, accounting for 40% of central nervous system tumors and 78% of adult neuromalignant tumors. In recent 30 years, although neuroimaging and glioma treatment have made great progress, the prognosis of malignant gliomas is still pessimistic. The 1-year and 5-year survival rates of adult malignant gliomas were about 30% and 13%, respectively. The median survival time of GBM was only about 12 months [2]. At present, gene therapy from theory to practice has brought great hope to thoroughly conquer the tumor and improve the prognosis of patients. However, there are still many links to be studied and broken down as soon as possible, among which the lack of efficient and stable carrier system and the particularity of blood-brain barrier structure in central nervous system are one of the important reasons leading to the inefficiency of its treatment. In this experiment, a two-step sol-gel method was used. The gelatin-siloxane nanoparticles (GS NPs),) were synthesized and a novel carrier system was constructed on the basis of which an aptamer TTAl and Fe304 were co-modified by polyethylene glycol (PEG), cationic polypeptide Tat, aptamer. It can not only penetrate the blood-brain barrier and specifically target brain tumor cells, but also have the effect of MRI imaging. The transfection efficiency of the plasmid DNA was investigated at the cell level to provide theoretical and practical support for the application of the nanoparticles as brain transporter. In the first part, the coupling of GS NPs and Fe304 was characterized by Fourier transform infrared spectroscopy (FT-IR). The physicochemical properties of the nanoparticles were studied by means of surface potential analysis, (Zeta potential), transmission electron microscope, (TEM), particle size analysis, (DLS), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). In the second part, laser confocal MRI and other imaging techniques were used to verify that GS NP-Fe3O4 as a gene vector could be effectively ingested by C6 cells, and GFP (green fluorescent protein) reporter gene could be efficiently expressed in tumor cells. Magnetic resonance imaging (MRI) was used to scan GS NP-Fe3O4 and Fe304 in a certain concentration range after cell coculture and the relaxation rate R2 was measured respectively to verify the possibility of GS NP-Fe3O4 as a contrast agent for MRI. In the third part, C6 glioma model was established by injecting C6 glioma cells under the guidance of stereotactic locator, and then the MRI imaging effects between different nanoparticles were compared by injecting GS NPs-Fe3O4 and Fe304, respectively through tail vein. Observe the change of signal. HE and Prussian blue staining were used to detect the pathological changes. By detecting rat C6 glioma slices and MRI imaging, this paper discusses whether GS NPs-Fe3O4 has the characteristics of specific targeted imaging, and can play a great advantage in tumor diagnosis, drug transport and targeted therapy.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
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