叶绿酸f光动力体外杀伤膀胱癌疗效及机制研究

发布时间：2018-10-16 09:22

【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。
[Abstract]:Objective photodynamic therapy is a new method of tumor therapy in recent years. Guang Min is the key of photodynamic therapy. The aim of this study was to determine the photodynamic effect and possible mechanism of chloric acid f on bladder cancer cells in vitro. Methods 5637 and T24 cells of bladder cancer were cultured in vitro. The growth inhibitory effect of folate f combined with 650nm laser on bladder cancer cells was detected by CCK-8 method. The distribution of subcells was observed by laser confocal microscopy after incubating with chloric acid f and specific mitochondrial fluorescence probe for 4 h, and the morphological changes of cancer cells 12 h after PDT staining by DAPI were observed under fluorescence microscope. Apoptosis rate was analyzed by flow cytometry. Results after 4J/cm2 laser irradiation, the growth inhibition rates of 5637 cells were 33.92% and 84.88%, respectively, which were 51.21% and 70.09% after 2J/cm2 laser irradiation, and 34.0360.11% and 86.51J / cm 2 for T24 cells with 2.55 渭 g/mL and 10 渭 g/mL concentrations, respectively, and 72.89% for T24 cells, and 86.51J / cm2 and 72.89cm ~ 2 for T24 cells, respectively.The inhibitory rates were 57.38% and 84.88%, respectively, for T24 cells, and for T24 cells, the inhibitory rates of 2.55 渭 g/mL and 10 渭 g/mL concentrations were 34.0360.11% and 86.51J / cm ~ 2, respectively, and 72.89%, respectively. Laser confocal microscopy showed that the red fluorescence of chloric acid f was consistent with the green fluorescence of mitochondrial probe, and both of them were in the cytoplasm. After DAPI staining, the cells in the treated group showed irregular nuclear edge, pyknosis, nucleolysis, fragmentation and increase of nucleosome fragments, but no such apoptosis was observed in the blank control group. The apoptosis rate of 5637 cells was (50.61 卤1.66)% in 4 渭 g/mL and (4.05 卤0.12)% in control group. The apoptosis rate of T24 cells was (54.3 卤1.32)% and (11.31 卤0.71)% in 4J/cm2 group. There was a significant difference between the two groups. Conclusion folic acid f combined with 650nm laser can effectively kill bladder cancer cells, and the killing effect is obviously increased with the increase of drug dose and laser energy. The mechanism of killing may be that the drug is located in mitochondria after entering cancer cells and induces apoptosis of tumor cells.
【作者单位】：上海市浦东新区公利医院泌尿外科;
【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)
【分类号】：R737.14

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