He Yu-Ying, Council Sarah E, Feng Li, Bonini Marcelo G, Chignell Colin F
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.
Photochem Photobiol. 2008 Jan-Feb;84(1):69-74. doi: 10.1111/j.1751-1097.2007.00199.x.
Singlet oxygen may be generated in cells by either endogenous or exogenous photosensitizers as a result of exposure to UV or visible irradiation. We have used immuno-spin trapping (Free Radic. Biol. Med. 36: 1214, 2004) to identify the subcellular targets of singlet oxygen generated by rose bengal (RB). Confocal fluorescence microscopy of HaCaT keratinocytes incubated with RB clearly showed that the dye entered the cells and was located mainly in the perinuclear region, probably associated with the Golgi apparatus and endoplasmic reticulum. Previous studies by Wright et al. (Free Radic. Biol. Med.34: 637, 2003) have shown that long-lived protein hydroperoxides (POOH) are present in cells exposed to singlet oxygen-generating dyes. The addition of reducing metal ions such as Cu+ to POOH results in the generation of protein-derived radicals, POO() and PO(), which react with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to give relatively stable spin adducts. In order to determine the subcellular localization of the protein-DMPO adducts, we exposed keratinocytes to RB/light exposure and then incubated the cells with Cu+ and DMPO. After staining with antibody against DMPO followed by a secondary Alexa Fluor 488 goat anti-rabbit IgG, the intracellular distribution of protein-DMPO adducts was determined by confocal microscopy. The subcellular localization of the protein DMPO adducts was coincident with that of RB. This approach may provide information on the spatial distribution of singlet oxygen generated in cells.
由于暴露于紫外线或可见光照射下，内源性或外源性光敏剂可能会在细胞中产生单线态氧。我们已使用免疫自旋捕获技术（《自由基生物学与医学》36: 1214, 2004）来鉴定孟加拉玫瑰红（RB）产生的单线态氧的亚细胞靶点。用RB孵育的HaCaT角质形成细胞的共聚焦荧光显微镜检查清楚地表明，该染料进入细胞并主要位于核周区域，可能与高尔基体和内质网相关。Wright等人先前的研究（《自由基生物学与医学》34: 637, 2003）表明，暴露于产生单线态氧的染料的细胞中存在长寿命蛋白质氢过氧化物（POOH）。向POOH中添加还原金属离子如Cu+会导致产生蛋白质衍生的自由基POO()和PO()，它们与自旋捕获剂5,5-二甲基-1-吡咯啉N-氧化物（DMPO）反应生成相对稳定的自旋加合物。为了确定蛋白质-DMPO加合物的亚细胞定位，我们将角质形成细胞暴露于RB/光照下，然后用Cu+和DMPO孵育细胞。在用抗DMPO抗体染色，然后用二级Alexa Fluor 488山羊抗兔IgG染色后，通过共聚焦显微镜确定蛋白质-DMPO加合物的细胞内分布。蛋白质DMPO加合物的亚细胞定位与RB的定位一致。这种方法可能提供有关细胞中产生的单线态氧的空间分布的信息。
