FRAP法对内源性GFP在活细胞中动态分布的共焦显微镜成像

各种分子在核质问的动态分布与它们的跨膜转运密切相关。离子、r证矾A和多数小分子量蛋白可以通过核孔复合物（NPG，nuclear pore complexes）在核质问自由扩散，而分子量大于70kDa的分子需要ATP和核定位序列才能实现跨膜转运。本实验利用荧光漂白后恢复（FRAP，fluorescence recovery after photobleaching）法观测人肺腺癌肿瘤细胞（ASTC-a-1）中表达的27 kDa EGFP在核质问的被动扩散，并以激光共焦显微镜进行实时成像。转染EGFP外源基因的肿瘤细胞系在经过半年的传代培养后仍能稳定而高效的表达其荧光标记。实验表明，EGFP分子可以通过核孔在核质间被动扩散，但扩散速度远低于在核内或质内的速度，没有证据表明EGFP可以在细胞问扩散。

Tracing the Dynamic Distribution of Endogenous Fluorescent Protein in the Tumor Cells by FRAP

The dynamic distribution of various molecules between nucleus and cytoplasm is concerned with their transfer cross the nuclear membrane in eukaryotic cells. Ions, mRNA and mostly smaller proteins are able to transfer through the NPC_S (nuclear pore complexes) by passive diffusion, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. We applied real-time image and fluorescence recovery after photobleaching (FRAP) to examine the nuclear pore permeability of 27 kDa EGFP in human lung cancer cells (ASTC-a-1) transfected with plasmid vector (pEGFP-C1). The transfected cell line expressed green fluorescent protein stably and highly effectively after passing on to new generations for more than half year. This endogenous fluorescent protein possesses many advantages and facilities as fluorescent tag instead of exogenous (fluorescence) dyes in fresh cell studies. We found that the EGFP molecules was able to diffuse freely through the NPC_S, but the diffusion rate was much lower than that in intra-nuclear or intra-cytoplasmic compartments, and no EGFP diffused passively between cell and cell.