叶绿酸f光动力体外杀伤膀胱癌疗效及机制研究

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650 nm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4 h后,激光共聚焦显微镜观察其亚细胞分布;DAPI染色PDT后12 h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 4 J/cm2激光照射后,1、2和4 μg/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 2 J/cm2激光照射后分别为25.4%、51.21%和 70.09%;对T24 细胞,2.5、5和10 μg/mL浓度药物4 J/cm2生长抑制率分别为 34.03%、60.11%和86.51%,2 J/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,4 μg/mL浓度结合4 J/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;10 μg/mL浓度结合4 J/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650 nm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.

Photodynamic effect of photosensitizer chlorophyllin f on human bladder cancer cells

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 T24 bladder 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-8 assay was used to measure the potocytotoxicity.Co-incubated with chlorophyllin f for 4 h, 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 T24 cells 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 T24 cells.In 5637 cells, with 4 J/cm2 light dose, the growth inhibition rates of 1、2 and 4 μg/mL chlorophyllin f were respectively 33.92%、57.38% and 84.88%;For T24 cells, with 4 J/cm2 light dose, the growth inhibition rates of 2.5、5 and 10 μg/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 featuresincluding 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 4 μg/mL and 4 J/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 10 μg/mL and 4 J/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 T24 cells, 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.