光化激活9-羟基脱镁叶绿酸-α诱导人喉鳞状细胞癌HN-3细胞凋亡与迁徙抑制  

Apoptosis and migration suppression of HN-3human laryngeal squamous cancer cells induced by photo-activation of 9-hydroxypheophorbide-α

作　　者：何培杰[1] 毛雯静 张焕康[1] 周梁[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院耳鼻咽喉-头颈外科上海200031

出　　处：《临床耳鼻咽喉头颈外科杂志》2015年第15期1367-1371,共5页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

摘　　要：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 effect and potential mechanisms about apoptosis induction and migration suppression of photodynamic therapy with a new photosensitizer,9-hydroxypheophorbide-α（9-HPbD）,and diode laser on HN-3human laryngeal squamous cancer cells.Method：The attached HN-3cancer cells were photosesitized with 0.29μg/ml and 0.59μg/ml 9-HPbD for 6hand irradiated by 664 nm diode laser for 15 min at an energy density of 2.0J/cm2 for activating 9-HPbD.Wound healing assay and photographing was respectively performed immediately after laser irradiation.Photographing focusing on the same location was repeated 12 h,24h and 36 hafter PDT and cells migration distance counted respectively.H2 DCFDA staining was used to assess accumulation of reactive oxygen series（ROS）1hafter PDT.MTT assay,Hoechst33342/PI double staining,western blotting were respectively performed to assess cellular viability,apoptosis and the expression of Enos,p-c-Jun,EGFR.Result：Phototoxicity and apoptosis on HN-3cells induced by 9-HPbD-PDT was exhibited in a dose-related manner.Neither 9-HPbD alone nor laser alone was cytotoxic to HN-3cells.Generation of ROS was initiated immediately after PDT.The apoptotic cells,marked with condensed/fragmented blue or pink nuclei,and up-regulated expression of eNOS,p-c-Jun were subsequently induced 24 hafter PDT.Coupled with a down-regulated expression of EGFR,aphotosensitizer dose-ralated cell migration suppression was initiated by PDT.After pretreatment of GSH or ascorbic acid,a kind of antioxidant,the efficacy of PDT-induced apoptosis and migration suppression was partially inhibited.Conclusion：Activation of p-c-Jun,eNOS and down-regulated expression of EGFR may respectively involve in the apoptosis induction and cell migration suppression after 9-HPbD-PDT.Generation of ROS may play an important role in the course of apoptosis induction and migration suppression of HN-3cells initiated by 9-HPbD-PDT.

关 键 词：喉肿瘤 鳞状细胞癌 光动力疗法 凋亡 迁徙运动 

分 类 号：R739.62[医药卫生—肿瘤]