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目的测定氯化锰(MnCl_2)处理脉络丛上皮细胞质Z310后升高的抑制素蛋白1(PHB1)在亚细胞中的分布及重定位,以及在细胞胞浆中的主要定位亚细胞器,并观察PHB1与细胞生长调控蛋白P53在细胞内是否有共定位关系。方法采用免疫蛋白印迹法(Western Blot)测定MnCl_2(0,100,200μmol/L)处理24 h后Z310细胞胞浆和胞核内的PHB1表达的变化及其重定位;采用免疫荧光法(Immunofluorescence,IF)结合激光共聚焦显微镜,检测并观察MnCl_2处理后PHB1的亚细胞空间分布,及其与P53的亚细胞共定位特征。结果 Western Blot检测表明,与对照组比较,MnCl_2处理后Z310细胞内PHB1的表达在胞浆中升高,同时在胞核内降低(P<0.05);免疫荧光法显示MnCl_2处理后PHB1在胞浆和胞核均存在,且在胞浆中主要分布于线粒体上;在胞浆和胞核内发现PHB1与P53有共定位(Co-localization)。结论MnCl_2处理后,上调表达的PHB1蛋白主要位于胞浆,同时发现胞核内PHB1降低,提示MnCl_2能够引起Z310细胞中PHB1蛋白升高并从胞核向胞浆移动,这种亚细胞水平的PHB1空间重定位(translocation)改变是锰毒性依赖性的;胞浆和胞核中的PHB1均与P53有共定位关系;胞浆中的PHB1主要存在于线粒体上。
OBJECTIVE: To determine the distribution and relocation of inhibin 1 (PHB1), a subcellular localization of organelles, in the cytoplasm of MnCl 2 -treated choroid plexus epithelial cytoplasm Z310 and observe the effects of PHB1 And cell growth regulatory protein P53 whether colocalization in the cell. Methods The changes of PHB1 expression in cytoplasm and nucleus of Z310 cells treated with MnCl 2 (0, 100, 200 μmol / L) for 24 h were determined by Western Blot. Immunofluorescence (IF) Laser confocal microscopy was used to detect and observe the subcellular distribution of PHB1 after MnCl 2 treatment and its co-localization with P53. Results The results of Western Blot showed that compared with the control group, the expression of PHB1 in Z310 cells was increased in the cytoplasm and decreased in the nucleus (P <0.05). The immunofluorescence showed that PHB1 in the cytoplasm And nucleus are present, and mainly distributed in the mitochondria in the cytoplasm; PHB1 and P53 found in the cytoplasm and nucleus Co-localization. Conclusions After treatment with MnCl 2, the up-regulated PHB1 protein mainly located in the cytoplasm, meanwhile, the decrease of PHB1 in the nucleus was found, suggesting that MnCl_2 could cause the PHB1 protein in Z310 cells to rise and move from the nucleus to the cytoplasm. The subcellular level of PHB1 Spatial translocation changes are manganese toxicity-dependent; PHB1 in both the cytoplasm and nucleus is colocalized with P53; and PHB1 in the cytoplasm is predominantly present in mitochondria.