摘要
目的:探讨光动力治疗(photodynamic therapy,PDT)对皮肤鳞状细胞癌的效应及细胞自噬在其中的作用。方法:在皮肤鳞状细胞癌细胞A431中,通过噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法检测5-氨基酮戊酸(5-aminolevulinic acid,ALA)-PDT对细胞增殖的影响,并进一步研究自噬抑制剂3-甲基嘌呤(3-methyladenine,3-MA)或自噬诱导剂5-氟尿嘧啶(5-fluorouracil,5-FU)联合ALA-PDT对细胞增殖的影响。Hoechst染色观察3-MA或5-FU联合ALA-PDT对A431细胞凋亡的影响,并用实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测凋亡相关基因如Bax、Bcl2、Survivin的表达情况。3-MA或5-FU联合ALA-PDT处理A431细胞后,通过RT-qPCR检测细胞中自噬微管相关蛋白1轻链3(autophagy microtubule associated protein 1 light chain 3,LC3)的表达水平及透射电镜来监测细胞自噬的发生。结果:ALA-PDT可抑制A431细胞的增殖并可诱导发生细胞自噬。进一步发现3-MA或5-FU联合ALA-PDT也可显著抑制皮肤鳞状细胞癌细胞的增殖。3-MA或5-FU联合ALA-PDT处理A431细胞后,与未处理组相比,Hoechst染色检测显示细胞凋亡被显著诱导;RT-qPCR检测显示LC3和Bax表达上调、Bcl2和Survivin表达下调;透射电镜显示细胞自噬被显著诱导,影响程度:5-FU+ALA-PDT>ALA-PDT>3-MA+ALA-PDT。结论:自噬调节可能是ALA-PDT治疗的关键点,ALA-PDT联合3-MA或5-FU可能成为治疗皮肤肿瘤,尤其是皮肤鳞状细胞癌的新策略。
Objective:To explore the effects of photodynamic therapy(ALA-PDT)on cutaneous squamous cell carcinoma(cSCC)and the role of autophagy on it.Methods:In human cSCC cell line A431,the effects of 5-aminolevulinic acid(ALA)-PDT on cell proliferation were detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay,and the effects of autophagy inhibitor 3-methyladenine(3-MA)or autophagy inducer 5-fluorouracil(5-FU)combined with ALA-PDT on cell proliferation were further studied.Hoechst staining was used to observe the effect of 3-MA or 5-FU combined with ALA-PDT on apoptosis of A431 cells,and real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of apoptosis-related genes such as Bax,Bcl2 and Survivin.After A431 cells were treated with 3-MA or 5-FU combined with ALA-PDT,the expression of autophagy microtubule associated protein 1 light chain 3(LC3)was detected by RT-qPCR and the autophagy was monitored by transmission electron microscopy.Results:ALA-PDT suppressed the proliferation but induced autophagy of A431 cells.Compared with untreated cells,ALA-PDT combined with 3-MA or 5-FU significantly induced apoptosis with hoechst staining,increased LC3 and Bax expression but decreased Bcl2 and Survivin expression with RT-qPCR measurement,promoted autophagy with transmission electron microscopy examination.Conclusion:Autophagy regulation may be a key point of ALA-PDT therapy.ALA-PDT combined with 3-FU or 5-FU may be a new strategy for treatment of skin cancers especially cSCC.
作者
张丽丽
季周婧
张捷
杨圣菊
ZHANG Lili;JI Zhoujing;ZHANG Jie;YANG Shengju(Department of Dermatology,the Affiliated Hospital of Nantong University,Nantong 226001)
出处
《南通大学学报（医学版）》
2020年第2期131-135,共5页
Journal of Nantong University(Medical sciences)
基金
南通市科技计划项目(MS12017015-7),南通大学附属医院“江海名医俊才计划”。
