光敏剂亚细胞分布位点光漂白作用的实验研究  

Photobleaching of Subcellular Distributed Sites of Photosensitizers:an Experimental Study

作　　者：戴维德[1,2] 李晓松[1,2] 曾晶[1,2] 刘凡光[1,2] 顾瑛[1,2] 

机构地区：[1]解放军总医院激光医学科,北京市100853 [2]现在卫生部北京医院超声科

出　　处：《中国激光医学杂志》2007年第3期137-141,共5页Chinese Journal of Laser Medicine & Surgery

基　　金：国家自然科学基金资助项目(60078021)

摘　　要：目的探讨光敏剂亚细胞分布位点光漂白作用的发生特点。方法将血卟啉单甲醚(hem atoprophyrin monomethyl ether,HMME)与传代培养ECV304细胞共同孵育12h后,采用荧光探针标记技术和细胞器-细胞荧光强度比值法对细胞内HMME进行亚细胞定位和定量分析。应用荧光显微镜汞灯照射以激发光敏剂产生光动力效应,并加入ROS探针H2DCF-DA检测单线态氧的产生。分别在光照前后采集四种细胞器及DCF的荧光图像并进行荧光强度的定量分析。结果细胞总荧光光强在光照后较光照前降低26.4%;以每种细胞器光照前的平均荧光光强比值为基准,则光照后线粒体、溶酶体、内质网和高尔基体分别降低了31.1%、23.4%、5.8%和5.0%。线粒体区域在单纯光照和有HMME存在情况下后均检测到DCF荧光的增强。结论光动力效应可能导致亚细胞结构内产生不同数量的单线态氧,会引起不同程度的光漂白作用,线粒体区域HMME光漂白速率较快;线粒体区域产生数量较多的单线态氧,可能是光动力效应的主要作用靶点之一。Objective To discuss the characters of photobleaching of subcellular sites of photosensitizers distributed in targeted cells. Methods ECV304 Cells were subeuhured and incubated with hematoporphyrin monomethyl ether （HMME） for 12 hours. Labeling technology of fluorescence probes and organelle-cell fluorescence intensity ratio analysis were adopted to study subcellular localization of HMME. Mercury lamp of fluorescence microscope was used as light source to have photosensitizers excitated and produce photodynamic effects. Fluorescence probe of ROS H2DCF-DA was used to detect production of sing, let oxygen. Fluorescence images of four organelles and DCF were recorded respectively before and after irradiation. Results Fluorescence intensity of the whole cell after irradiation was decreased by 26.4%. According to every organelles, the average fluorescence intensity ratio of the mitochondria, lysosome, ER and Golgi were decreased by 31.1%, 23.4% ,5. 8% and 5.0% after irradiation separately. Enhancing fluorescence of DCF was detected in mitochondria with HMME or after irradiation. Conclusions Photodynamic effects may induce various quantity of singlet oxygen in various organelles and bring various degree of photobleaching. The speed of photobleaching of HMME in mitochondria is faster than others ; more singlet oxygen is produced in mitochondria, which maybe one of primary targets of photodynamic therapy.

关 键 词：光敏剂 细胞内分布 光漂白 

分 类 号：Q2-33[生物学—细胞生物学]