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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 Photodynamic therapy is a newly emerging cancer treatment in recent years. Photosensitizers are the key to photodynamic therapy. This study aimed to clarify the effect of chlorophyll f photodynamic therapy on bladder cancer cells in vitro and its possible mechanism. Methods Bladder cancer 5637 and T24 cells were cultured in vitro. The growth inhibition of bladder cancer cells was detected by CCK-8 assay. After incubating cells with fluorescein and specific mitochondrial fluorescence probe for 4 hours, the distribution of subcellular distribution was observed by confocal laser scanning microscope. The morphological changes of cells were observed under fluorescence microscope 12 hours after DAPI staining. Flow cytometry The rate of apoptosis was analyzed. Results After 4J / cm2 laser irradiation, the inhibition rates of chlorophyll f at 1, 2 and 4μg / mL on 5637 cells were 33.92%, 57.38% and 84.88%, respectively, 25.4% and 51.21% after 2J / cm2 laser irradiation % And 70.09%, respectively. The growth inhibition rate of T24 cells at doses of 2.5, 5 and 10μg / mL was 34.03%, 60.11% and 86.51%, respectively, and the rate of 2J / cm2 was 31.79%, 53.83% and 72.89%, respectively. Laser confocal microscopy showed that the red fluorescence of chlorophyll f was consistent with the distribution of green fluorescence of mitochondrial probe, both in the cytoplasm and no distribution in the nucleus. After DAPI staining, the cells in the treated group showed irregular nuclei, nuclear pyknosis, nuclear lysis, fragmentation and increase of nucleosome fragments. The apoptotic cells were not observed in the blank control group. The results of flow cytometry showed that the apoptosis rate of 5637 cells treated with 4μg / mL laser combined with 4J / cm2 laser was (50.61 ± 1.66)% and that of control group was (4.05 ± 0.12)%, the difference was statistically significant , U = 6.21, P = 0.042. The apoptosis rate of T24 cells treated with 10μg / mL and 4J / cm2 group was (54.3 ± 1.32)% and that of the control group was (11.31 ± 0.71)%. The difference was significant, U = 7.35, P = 0.030. Conclusion Chlorophyllin f combined with 650nm laser can effectively kill bladder cancer cells. The killing effect is obviously increased with the increase of dose and laser energy. Killing mechanism may be the drug enters the cancer cells located in the mitochondria, induce tumor cell apoptosis.