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

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 feathers could not be found in the control cells.The apoptosis rate of the 5637 cell panel 4μg/mL and 4J/cm2 light dose was（50.61±1.66）%,that of the control panel was（4.05±0.12）%,the difference of apoptosis rate is statically significant（U=6.21,P=0.042）.With 10μg/mL and 4J/cm2 light dose,the apoptosis rate of T24 cells was（54.3±1.32）%,and that of the control panel was（11.31±0.71）%,the difference of apoptosis rate is statically significant（U=7.35,P=0.030）.CONCLUSIONS The chlorophyllin f（combined with 650 nm laser light）can efficiently kill both 5637 and T24cells,the cell inhibition rate correlates with the concentration of chlorophyllin f and the laser dose.Chlorophyllin f could locate in the mitochondria.Chlorophyllin f-mediated-PDT may induce apoptosis which may be one of the mechanisms against bladder cancer cells.