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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.

利用膜渗透性肽载体的细胞内递送是最近开发的方法，其已成功地用于将各种生物活性分子转运到细胞中以改变细胞功能。本研究的目的是使用荧光显微镜观察内化的肽载体在真实的时间，并分析细胞内的靶向信号，包括那些为核和线粒体的肽载体的连接对其细胞定位的影响。细胞的共聚焦显微镜观察显示，大多数肽载体被细胞通过内吞作用摄取，但由于肽在胞质溶胶中的高积累，难以分析扩散到胞质溶胶中的肽载体。然而，在不含血清的培养基中或在存在高浓度载体的情况下，观察到肽载体的有效胞质扩散。将SV40核定位信号肽连接到载体上成功地增强了缀合物在细胞核中的积累，但通过连接线粒体定位信号没有观察到显著的效果。因此，我们得出结论，线粒体定位信号肽可能是有效的，只有新的细胞内产生的蛋白质，但不为细胞内交付的蛋白质。这些发现对细胞内靶向载体的研究具有一定的参考价值。