血卟啉单甲醚在鼠肺毛细血管内皮细胞内的亚细胞定位

目的 :研究血卟啉单甲醚 (HMME)在鼠肺毛细血管内皮细胞内的亚细胞定位 ;方法 :孵育时间分别为 2小时、12小时、2 4小时 ,四种荧光探针标记四种细胞器。倒置荧光显微镜和致冷CCD探测HMME和探针荧光图像。定性比较两种荧光分布模式 ,并定量计算高探针荧光区域与细胞内HMME荧光均量比I2 /I1;结果 :三种孵育时间下 ,HMME荧光均呈胞浆弥散分布 ,且在核周附近的高尔基体灰度较高 ,细胞核几乎无荧光分布。I2 /I1高低顺序 2小时组 :高尔基体 >内质网 >线粒体 /溶酶体 ;12小时组 :高尔基体 >溶酶体 >内质网 /线粒体 ,且内质网组显著下降与高尔基体组略有下降伴随溶酶体组显著上升 ;2 4小时组 :高尔基体 >线粒体 /内质网 >溶酶体 ,且溶酶体组显著下降与高尔基体组一定下降伴随线粒体组与内质网组显著上升 ;结论 :倒置荧光显微镜和致冷CCD能有效应用于光敏剂亚细胞定位研究 ,定量分析方法对弥散分布光敏剂尤其适合 ;三种孵育时间下 ,高尔基体为HMME主要定位点 ,细胞核几乎无分布。短孵育时间 ,内质网有一定分布 ;中孵育时间 ,由内质网(主要 )和高尔基体到溶酶体再分布 ;长孵育时间 ,由溶酶体和高尔基体到线粒体和内质网再分布。

Subcellular Localization of HMME in Murine Lung Endothelial Cells

Objective: To study HMME subcellular localization in murine lung endothelial cells; Methods: The incubation time was 2 hours, 12 hours and 24 hours, respectively. Four fluorescent probes were used to label four subcellular organelles. The fluorescence images of HMME and probes were detected by using the inverted fluorescence microscope and cooled CCD. The two patterns of fluorescence distribution were compared qualitatively, and I2/I1 was calculated quantitatively, which represented the ratio of HMME fluorescence intensities in area with high probe fluorescence and the cell; Results: For three incubation times, HMME fluorescence has diffuse cytoplasmic distribution, with little nuclear stain and brighter perinuclear area in Golgi apparatus. The order of I2/I1 in 2 hours group is: Golgi>endoplasmic reticulum(ER)>mitochondria/lysosome; in 12 hours group it is: Golgi>lysosome>ER/mitochondria, with obvious decrease in ER group and a little decrease in Golgi group as well as pronounced increase in lysosome group; in 24 hours group it is: Golgi>mitochondria/ER>lysosome, with obvious decrease in lysosome group and some decrease in Golgi group as well as pronounced increase in mitochondria and ER groups. Conclusion: The inverted fluorescence microscope and cooled CCD can be effectively used for studying the subcellular localization of photosensitizers, and the quantitative analysis method is especially suitable to those with diffuse distribution; For three incubation times, Golgi is the prominent localization site of HMME with no nuclear stain. For short incubation time, ER exhibits some labeling; For middle one, some relocalization from ER (mainly) and Golgi to lysosome occurs; For long one, some relocalization from lysosome and Golgi to mitochondria and ER occurs.