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人类抗原R(human antigen R,HuR)是一种多功能RNA结合蛋白,参与细胞应激颗粒(stress granules,SGs)的构成。SGs是细胞在受到外界环境刺激时在胞浆中形成的颗粒状结构。该研究是利用光漂白荧光损失(fl uorescence loss in photobleaching,FLIP)技术对活细胞内的HuR蛋白颗粒进行应激动力学分析。首先,利用脂质体将RFP-HuR重组质粒瞬时转染入HeLa细胞,以Western blot和细胞免疫荧光实验确定是否实现对于HuR蛋白的红色荧光蛋白(red fl uorecent protein,RFP)标记;然后以405 nm激光束脉冲式重复光漂白HuR应激颗粒,分别监测同一漂白细胞内的其他HuR颗粒以及核内荧光信号,并以邻近的未漂白细胞作为对照组。实验结果表明,转染重组质粒后可有效表达RFP-HuR融合蛋白,且与SGs标记蛋白G3BP存在共定位关系。在第一个光漂白循环,漂白区荧光密度便从2 500 a.u.降低至0 a.u.;而经过约12个漂白循环(240 s)后,邻近HuR颗粒的荧光密度从漂白前的1 800 a.u.左右降低并维持在200 a.u.左右,表明活细胞内的HuR颗粒呈现高度的动态性;而胞核区荧光密度亦从4 400 a.u.降低至2 000 a.u.左右,表明HuR蛋白是一种核浆穿梭蛋白,在SGs、胞浆及胞核之间存在一定的动态平衡。利用FLIP技术可以分析并比较SGs不同成分的应激动力学属性,有助于进行SGs相关临床疾病的分子机制探讨。
Human antigen R (HuR) is a multifunctional RNA-binding protein involved in the formation of stress granules (SGs). SGs are the granular structures formed in the cytoplasm when cells are stimulated by the external environment. This study used the fl uorescence loss in photobleaching (FLIP) technique to perform stress-kinetic analysis of HuR protein particles in living cells. Firstly, the recombinant plasmids of RFP-HuR were transiently transfected into HeLa cells by lipofectamine. Western blot and immunofluorescence assay were used to determine whether the red fluorescent protein (RFP) tag of HuR protein was achieved. nm laser beam were used to photobleach HuR stress particles to monitor the other HuR particles in the same bleached cells and the fluorescence signal in the nucleus respectively. The adjacent unbleached cells were used as the control group. The experimental results showed that RFP-HuR fusion protein was efficiently expressed after transfected with recombinant plasmid, and co-localized with SG3 marker protein G3BP. At the first photobleaching cycle, the fluorescence density in the bleaching zone was reduced from 2,500 au to 0 au. After about 12 bleaching cycles (240 s), the fluorescence densities of neighboring HuR particles decreased from around 1,800 au before bleaching And remained at about 200 au, indicating that the HuR particles in living cells showed a high degree of dynamic; while the fluorescence density in the nucleus also decreased from 4 400 au to about 2 000 au, indicating that HuR protein is a nuclear plasma shuttle protein. SGs, there is a certain dynamic balance between cytoplasm and nucleus. The use of FLIP technology can analyze and compare the different components of SGs stress kinetic properties, contribute to the molecular mechanisms of SGs-related clinical disease.