Development of intracellular targeting peptide vectors and the real-time observation in cells.

细胞内靶向肽载体的研制及细胞内实时观察。

基本信息

批准号：
17390029
负责人：
FUTAKI Shiroh
金额：
$ 9.73万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390029/
关键词：
arginine-rich peptide intracellular delivery peptide vector endocytosis nucleus mitochondria

项目摘要

Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has been employed successfully to transport various bioactive molecules into cells to modify cell functions. The purpose of this study is to observe internalized peptide vectors in real time using fluorescence microscopy and to analyze the effect of the attachment of intracellular targeting signals including those for nucleus and mitochondria to the peptide vectors on their cellular localization. Confocal microscopic observation of the cells revealed that most of the peptide vectors were taken up by the cells by endocytosis, but it was difficult to analyze the peptide vectors diffusing into cytosol due to the high accumulation of the peptides in cytosol. However, in culture medium without containing serum or in the presence of high concentration of the vectors, effective cytosolic diffusion of the peptide vectors was observed. Attachment of the SV40 nuclear localization signal peptide to the vectors successfully enhanced the accumulation of the conjugates into nucleus, but no significant effect was observed by the attachment of mitochondrial localization signals. We thus concluded that mitochondria localization signal peptides may be effective only for newly in-cell generated proteins but not for intracellularly delivered proteins. These findings should be useful for the intracellular targeting vectors.

使用膜可渗透肽载体的细胞内递送是一种最近开发的方法，已成功地将各种生物活性分子转运到细胞中以修饰细胞功能。这项研究的目的是使用荧光显微镜实时观察内部化的肽载体，并分析细胞内靶向信号的附着在其细胞定位上的附着在细胞核和线粒体上的效果。对细胞的共聚焦显微镜观察表明，大多数肽载体被细胞通过内吞作用吸收，但是由于细胞质中肽的高积累，很难分析肽载体扩散到胞质醇中。但是，在不包含血清或高浓度向量的培养基中，观察到肽载体的有效胞质扩散。 SV40核定位的附着信号肽在向量上成功增强了共轭物的积累，但是通过线粒体定位信号的附着，没有观察到显着影响。因此，我们得出的结论是，线粒体定位信号肽可能仅对新近的内部产生的蛋白质有效，而不是细胞内递送的蛋白质。这些发现对于细胞内靶向向量应该有用。