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目的:探讨新型光敏剂9-羟基脱镁叶绿酸-α(9-HPbD)介导的光动力疗法(PDT)对HN-3人喉鳞状细胞癌细胞的凋亡诱导、迁徙抑制作用及其机制。方法:以不同浓度的9-HPbD(0.29μg/ml,0.59μg/ml)孵育贴壁的HN-3细胞6h,664nm二极管激光(能量密度为2.0J/cm2)照射光敏化的HN-3细胞15min。光照后立即行细胞划痕,并于光照后0、12、24、36h分别在相同划痕位置进行拍照,计算细胞迁徙距离;光照后1h行H2DCFDA染色并分别以荧光法及流式细胞术测定活性氧生成(ROS);光照24h后行MTT实验、Hoechst33342/PI双重染色、蛋白印迹法(Western blotting)评价细胞增殖活力、细胞核形态及eNOS、p-c-Jun、EGFR表达。结果:19-HPbD-PDT显著抑制HN-3细胞增殖,9-HPbD单独孵育(未进行激光照射)与单纯激光照射均未显示对HN-3细胞的生长抑制。2PDT后HN-3细胞活性氧产生明显增加,细胞核浓缩、碎裂,eNOS、p-c-Jun表达上调。39-HPbD-PDT显著抑制HN-3细胞迁徙能力,呈光敏剂剂量相关性。PDT后HN-3细胞EGFR表达下调。4以活性氧阻滞剂谷胱甘肽(GSH)、抗坏血酸预孵育HN-3细胞,光动力触发的凋亡诱导、迁徙抑制等被部分阻滞。结论:19-HPbD-PDT对HN-3细胞具有光化学毒性,p-c-Jun通路激活及eNOS表达上调、EGFR表达下调可能参与了细胞凋亡与迁徙抑制的诱导。2活性氧生成在9-HPbD-PDT诱导HN-3细胞凋亡与迁徙抑制中发挥重要作用。
OBJECTIVE: To investigate the apoptosis-inducing effect of 9-hydroxy-pheophorbide-α (9-HPbD) -mediated photodynamic therapy (PDT) on HN-3 human laryngeal squamous cell carcinoma Its mechanism. METHODS: The adherent HN-3 cells were incubated with different concentrations of 9-HPbD (0.29μg / ml, 0.59μg / ml) for 6h. The sensitized HN-3 cells were irradiated with 664nm diode laser (energy density 2.0J / cm2) 15min. The cells were scratched immediately after illumination and taken at 0, 12, 24, and 36 hours after illumination respectively at the same position of scratches to calculate the migration distance of cells. H2DCFDA staining was performed 1 h after the light exposure and the fluorescence staining and flow cytometry The activity of reactive oxygen species (ROS) was measured. MTT assay, Hoechst33342 / PI double staining and Western blotting were used to evaluate the cell viability, nuclear morphology and the expression of eNOS, pc-Jun and EGFR. Results: The proliferation of HN-3 cells was significantly inhibited by 19-HPbD-PDT. The inhibition of HN-3 cells growth by 9-HPbD alone (without laser irradiation) and laser irradiation alone were not observed. After 2PDT, reactive oxygen species (ROS) production of HN-3 cells increased significantly, and nuclear condensation, fragmentation, eNOS and p-c-Jun expression were up-regulated. 39-HPbD-PDT significantly inhibited the migration ability of HN-3 cells in dose-dependent manner. After PDT HN-3 cells EGFR expression down. 4 was blocked by glutathione (GSH), ascorbic acid pre-incubated HN-3 cells, photodynamic induced apoptosis induction, migration inhibition and so on. Conclusion: 19-HPbD-PDT has photochemical toxicity on HN-3 cells, activation of p-c-Jun pathway and upregulation of eNOS. The downregulation of EGFR may be involved in the induction of apoptosis and migration inhibition. 2 ROS generation plays an important role in 9-HPbD-PDT induced HN-3 cell apoptosis and migration inhibition.