叶绿酸f光动力体外杀伤膀胱癌疗效及机制研究  

Photodynamic effect of photosensitizer chlorophyllin f on human bladder cancer cells

作　　者：郑景存[1] 胡传义[1] 姜宁[1] 刘爽[1] 王国增[1] 

机构地区：[1]上海市浦东新区公利医院泌尿外科,上海200135

出　　处：《中华肿瘤防治杂志》2017年第8期529-534,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81272845);上海市浦东新区科技发展基金(PKJ2015-Y21);上海市卫计委重点学科资助(ZK2015A11)

摘　　要：目的光动力治疗是近年来新兴起的一种肿瘤治疗方式,光敏剂是光动力治疗的关键。本研究旨在明确叶绿酸f光动力学体外杀伤膀胱癌细胞的效果及其可能机制。方法体外培养膀胱癌5637和T24细胞,CCK-8法检测叶绿酸f结合650nm激光对膀胱癌细胞的生长抑制作用。叶绿酸f和特异性线粒体荧光探针同细胞共孵育4h后,激光共聚焦显微镜观察其亚细胞分布;DAPI染色PDT后12h的癌细胞,荧光显微镜下观察细胞形态学改变;采用流式细胞仪分析凋亡率。结果 4J/cm2激光照射后,1、2和4μg/mL的叶绿酸f对5637细胞生长抑制率分别为33.92%、57.38%和84.88%,在2J/cm2激光照射后分别为25.4%、51.21%和70.09%;对T24细胞,2.5、5和10μg/mL浓度药物4J/cm2生长抑制率分别为34.03%、60.11%和86.51%,2J/cm2分别为31.79%、53.83%和72.89%。激光共聚焦显微镜观察到叶绿酸f的红色荧光与线粒体探针的绿色荧光分布范围一致,都在细胞质中,细胞核中无分布。DAPI染色后荧光显微镜下可见处理组细胞均出现胞核边缘不规则,核固缩,核溶解、碎裂,核小体碎片增加等现象,空白对照组细胞则未见到此类凋亡表现。流式细胞术检测结果显示,4μg/mL浓度结合4J/cm2激光处理组5637细胞凋亡率为(50.61±1.66)%,对照组凋亡率为(4.05±0.12)%,差异有统计学意义,U=6.21,P=0.042;10μg/mL浓度结合4J/cm2组T24细胞凋亡率为(54.3±1.32)%,对照组凋亡率为(11.31±0.71)%。差异有显著性意义,U=7.35,P=0.030。结论叶绿酸f结合650nm激光,能够高效杀灭膀胱癌细胞,该杀灭效应随药物剂量及激光能量的增加明显升高。杀灭机制可能是药物进入癌细胞后定位于线粒体,诱导肿瘤细胞凋亡。OBJECTIVE We synthesized a new and low-cost photosensitizer（PS）,chlorophyllin f,and investigated its photodynamic effects and potential mechanisms of phototoxicity in human bladder cancer cells.METHODS 5637 and T24bladder cancer cells were cultivated in vitro.Different concentrations of chlorophyllin f were added to the cells and then irradiated by 650 nm laser light.Controls panels are：cells in cubated with chlorophyllin f without laser light;cells exposed to light without chlorophyllin f as well as both blank（cells with neither laser light nor chlorophyllin f）.The Cell Counting Kit-8assay was used to measure the potocytotoxicity.Co-incubated with chlorophyllin f for 4h,Mito-Tracker Green probe was used to label mitochondria.To reveal chlorophyllin f intracellular localization,CLSM was used to excite the fluorescent light of chlorophyllin f and the probe by both 400 nm and 488 nm channels.The morphological changes of the f-PDT-treated 5637 and T24cells after DAPI staining were observed under fluorescence microscopy.After chlorophyllin f-mediated-PDT,flow cytometry was used to analyze the apoptosis rates of cells.RESULTS Chlorophyllin f can effectively kill 5637 and T24cells.In 5637 cells,with 4J/cm2 light dose,the growth inhibition rates of 1、2and 4μg/mL chlorophyllin f were respectively 33.92%、57.38%and 84.88%;For T24 cells,with 4J/cm2 light dose,the growth inhibition rates of 2.5、5and 10μg/mL chlorophyllin were 34.03%、60.11% and 86.51%,respectively.The control panels（chlorophyllin f or laser light alone）did not show cytotoxicity.The outcomes of CLSM revealed the red fluorescence light emitted by chlorophyllin f and green fluorescence emitted by Mito-Tracker Green probe distributed almost the same range.Both the cells which were treated by f-PDT and stained by DAPI,revealed the typical apoptotic morphological features including nuclear with irregular edges,karyopycnosis,nuclear dissolution,nuclear fragmentation,increased nucleosomes and other debris under fluorescence microscope;these fe

关 键 词：膀胱癌 叶绿酸f 光动力学 线粒体 凋亡 

分 类 号：R737.14[医药卫生—肿瘤]