共焦荧光显微定量研究血卟啉单甲醚亚细胞定位

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、2 4小时 ,四种荧光探针标记四种细胞器。激光扫描共聚焦显微镜 (LSCM)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 :两种孵育时间下 ,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 >溶酶体 线粒体 ;2 4小时组 :高尔基体 >线粒体 内质网 >溶酶体 ,且高尔基体组显著下降伴随线粒体组大幅上升 ;结论 :此定量分析方法能有效用于光敏剂亚细胞定位研究 ,对弥散分布光敏剂尤其适合 ;两种孵育时间下 ,高尔基体为HMME主要定位点 ,内质网有一定分布 ,溶酶体分布很少 ,细胞核几乎无分布 ;短孵育时间线粒体分布很少 ,长孵育时间由高尔基体到线粒体有一定再分布

Quantitative study of HMME subcellular localization via confocal fluorescence microscopy

Objective:To study HMME subcellular localization in murine lung endothelial cells;Methods:The incubation time is 2 hours and 24 hours,respectively.Four fluorescent probes are used to label four subcellular organelles.The fluorescence images of HMME and probes are detected by LSCM.The two patterns of fluorescence distribution were compared qualitatively,and I 2/I 1 is calculated quantitatively,which represents the ratio of HMME fluorescence intensities in area with high probe fluorescence and the cell;Results:For both incubation times,HMME fluorescence has diffuse cytoplasmic distribution,with little nuclear stain and brighter perinuclear area in Golgi apparatus.The order of I 2/I 1 in 2 hours group is :Golgi>endoplasmic reticulum(ER)>lysosome/mitochondria;in 24 hours group is :Golgi>mitochondria/ER>lysosme,with obvious decrease in Golgi group and pronounced increase in mitochondria group.Conclusions:The quantitative analysis method can be effectively used for studying the subcellular localization of photosensitizers,especially suitable to those with diffuse distribution;For both incubation times,Golgi is the prominent localization site of HMME,and ER exhibits some labeling with a little lysosome and no nuclear stain.For short incubation time,mitochnodria has a little staining;For long one ,some relocalization from Golgi to mitochondria occurs.