【作者】 程利容；

【导师】 刘祖黎；

【作者基本信息】 华中科技大学 ， 材料物理与化学， 2007， 硕士

【摘要】 近年来,由于磁性纳米材料性能稳定,较易制备,可与多种分子复合使粒子表面功能化,并且它具有超顺磁性,为样品的分离、富集和提纯提供了很大的方便,从而使它逐渐成为新一代的标记物而广泛用于生物化学、分子生物学和临床医学等多个领域,如生物学分离、细胞标记、药物传送、免疫检测和磁共振造影剂等方面。其中,磁性细胞标记实质上是以磁共振对比剂为基础的,因为磁共振成像已经作为体内细胞研究的一种潜在手段。应用磁共振成像可动态观察干细胞和其他细胞的迁移,在转染剂的作用下,可使超顺磁性氧化铁颗粒进入细胞,从而可在活体下动态观察细胞的行踪。现如今核磁共振成像已经可以提供磁性颗粒在体外、动物和人体内的空间成像位置:研究者将磁共振对比剂——超顺磁性氧化铁纳米颗粒(SPION)在病理学上用于探测肝、脾、和淋巴结。经过噬菌作用后,自旋-自旋弛豫时间T2缩短,磁共振信号弱,目标组织很容易被检测。注入SPION之后,目标组织中的磁性颗粒加速而且会滞留一段时间,刚好提供一个间隙来确定颗粒的位置。而磁性细胞标记不仅仅需要提供标记后的细胞的成像位置,还需要提供细胞周围的环境来从而获得细胞中重要生物分子的位置、运动及其他分子的相互作用等。更重要的是,用于磁性细胞标记的标记物是需要能进入细胞的。这里我们利用了HIV-1 Tat的强穿透力:它能将与之连接的多肽、蛋白质及DNA以一种浓度依赖的方式高效快速的导入细胞内,而细胞的正常结构和功能不受影响。在本文中,我们应用化学方法将HIV-1 Tat蛋白转导应用于细胞的磁性荧光标记物的制备研究。该方法制得的标记物可以顺利进入细胞,既具有超顺磁性,可以进行核磁共振成像,又发荧光,可进行活体光学成像。实验以磁共振对比剂——超顺磁性氧化铁纳米粒子(SPION)为核心,在其表面连上氨基官能团,然后通过交联剂的中介作用,连上带荧光的Tat蛋白。在这一连串的实验中,每一步实验的检测都是必要的。通过一些物理和化学检测我们证实了这些实验过程,从而最终制得的带荧光的超顺磁性纳米颗粒可以作为生物医学上的荧光磁性标记物来应用。更多还原

【Abstract】 In recent yeas, because of its stabillity, facility prepareation, and being prone to combine various molecules to function its surface, and because of its superparamagnetic and being convenient to separate, collect and purify sample, nanoparticles have been wildly applied to many field in biochemistry, molecular biology and clinical medicine as marker of the new generation such as magnetic separation, cell marking , medicine transmit , immune detecting and contrast agent of magnetic resonance imaging (MRI). Among them, cell markers applied in cell marking are based on contrast agent of MRI in fact, because magnetic resonance imaging has become a potential study of cell in vivo image. We observe dynamic migration stem cell and other cells by MRI. Superparamagnetic iron oxide enters the cell under the function of dye agent transferred, and then we could observe where cell goes in vivo.SPION (superparamagnetic iron oxide nanoparticles) are well established as magnetic resonance(MR) reporters for detecting pathologies in the liver, spleen and lymph nodes where, after phagocytosis, their presence is evident primarily by a darkening effect on T2 weighted images .After injection, magnetic nanoparticles accumulate in target tissues and are degraded over a period of several days, providing a convenient interval to determine nanoparticle disposition, MRI is now capable of providing high spatial resolution images of nanoparticles disposition in vitro, in small animals, and in human.In cell marking, the markers should provide not only the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, but also the information on their environment by optical imaging techniques. Here we made the best of HIV-1 Tat in strong penetrability: It could lead peptide, protein and DNA which is linked in cell with high efficiency depending on concentration, while regular structure and function of cells are not affected.In this communication, we adopted chemical reaction to prepare magnetic and fluorescent markers in which HIV-1 Tat (Trans-activating transcriptional activator) PTD (protain transduction domain) was applied. The marker could smoothly enter the cells. They may be applied to MRI because of the superparamagnetism and optical imaging in vivo because of the luminescence. The experiment take magnetic resonance contrast agent-----superparamagnetic iron oxide nanoparticles (SPION) as the core, amino groups conjugated to its surface continually, and then conjugate to Tat protein through crosslinking intermediary. In above experiments, the testing of each step is all necessary. We confirmed these experimental processes by some physics and chemistry tests, so the last samples are allowed to apply in biomedicine as biomarkers.更多还原