基于原子力显微镜单分子力谱技术研究枝状聚合物聚酰胺胺的跨膜动态过程

发布时间：2018-05-17 22:15

本文选题：原子力显微镜 + 单分子力示踪　；参考：《长春工业大学》2017年硕士论文

【摘要】：相比于众多传统的高分子药物载体,树枝状大分子聚合物聚酰胺胺(PAMAM)具有很多特有的优势,它的表面连有大量的官能团,经过修饰可以连接更多的药物分子;其次,在空间上呈球状分布的高代数树枝状PAMAM大分子,内部存在着较大的孔腔,这些孔腔可以包埋药物分子。因此越来越多的研究人员着手研究PAMAM作为药物载体的应用,然而由于研究手段的空间和时间分辨率限制,作为药物载体进入细胞的第一步——跨膜过程并不是很清楚。在此,我们利用基于原子力显微镜(AFM)的单分子力示踪技术(可以测量皮牛级的力和微秒量级的动态过程)对单个第五代树枝状化合物分子(G5-PAMAM)进入细胞的跨膜动态过程进行研究。我们实时记录了单个PAMAM进入活细胞的动态过程,测量到跨膜力的大小及其进入细胞所需的时间,并研究了其进入细胞的内吞机制。本研究将为PAMAM作为药物载体的应用提供更多的理论基础,对生物医学领域有重要意义。研究取得进展如下:1.本研究记录了单个G5-PAMAM进入活细胞的动态过程,测量到跨膜力的大小及其进入细胞所需要的时间。G5-PAMAM纳米粒子与细胞作用的内吞力的范围是50pN至425 pN,其平均的作用力是172±74 pN,进入细胞内所需时间的范围是2.5 ms至65 ms,平均值为19±11 ms。2.另外,本研究还揭示了G5-PAMAM进入细胞的内吞机制。不仅用力示踪技术在单分子水平上做了对照和阻碍实验,而且也通过激光共聚焦显微镜量化了相对于对照实验(未用阻碍试剂处理)而言用阻碍试剂预处理后的细胞内G5-PAMAM粒子的平均荧光强度。值得注意的是,两种实验方法得出的结论一致。非洲绿猴肾细胞(Vero)内吞G5-PAMAM通过小窝蛋白和网格蛋白介导的内吞及巨胞饮方式(网格蛋白及巨胞饮为主)完成,其中网格蛋白介导和巨胞饮是主要的途径。
[Abstract]:Compared with many traditional polymer drug carriers, the dendritic macromolecular polymer PAMAM has many unique advantages. It has a large number of functional groups attached to its surface, which can be modified to connect more drug molecules. High algebraic dendritic PAMAM macromolecules with spherical distribution in space have large pore cavities which can be encapsulated into drug molecules. Therefore, more and more researchers have begun to study the application of PAMAM as drug carrier. However, due to the limitation of space and time resolution, the first step-transmembrane process of drug carrier entering cells is not very clear. In this paper, the transmembrane dynamic process of single fifth generation dendrimer (G5-PAMAM) into cells was studied by using AFM-based single molecular force tracer technique (which can measure the dynamic processes of skin cattle force and microsecond order of magnitude). We recorded the dynamic process of a single PAMAM entering living cells in real time, measured the transmembrane force and the time it took to enter the cells, and studied the endocytosis mechanism. This study will provide more theoretical basis for the application of PAMAM as a drug carrier, and will be of great significance to the biomedical field. The progress of the study is as follows: 1. In this study, the dynamic process of single G5-PAMAM entering living cells was recorded. The transmembrane force and the time required to enter the cell. The endocytosis range of G5-PAMAM nanoparticles was from 50pN to 425pN, the average force was 172 卤74 PN, and the time to enter the cell was 2.5 Ms to 65 Ms, with an average of 19 卤11 ms.2. In addition, this study also revealed the endocytosis mechanism of G5-PAMAM entering cells. Not only were forced tracer techniques used to control and block experiments at the single molecular level, The average fluorescence intensity of G5-PAMAM particles pretreated with hindrance reagent was quantified by laser confocal microscopy. It is worth noting that the two experimental methods come to the same conclusion. The endocytosis of G5-PAMAM in the kidney cells of African green monkey is accomplished by the way of endocytosis and giant cell drink (mainly grid protein and giant cell drink) mediated by nest protein and grid protein, among which grid protein and giant cell drink are the main pathway.
【学位授予单位】：长春工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ460.1;O633.22

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