摘要
目的探讨5-氨基酮戊酸光动力疗法(ALA-PDT)联合3-甲基腺嘌呤(3-MA)对皮肤鳞状细胞癌A431细胞的杀伤作用及其机制研究。方法体外培养人皮肤鳞状细胞癌A431细胞,设置不同的ALA浓度(0、0.20、0.40、0.80、1.60 mmol/L)及不同的培养时间(12、24、48 h),对A431细胞进行ALA-PDT处理,采用MTT法检测细胞增殖活性并计算半抑制浓度(IC50)值。根据实验需要将A431细胞分为空白对照组、ALA-PDT组、3-MA组和ALA-PDT+3-MA组。分组处理后,MTT法检测细胞增殖;流式细胞术分别检测细胞凋亡(Annexin V-FITC/PI双染法)以及细胞线粒体膜电位变化(JC-1染色法);单丹磺酰尸胺(MDC)染色法观察细胞自噬情况;Western blot检测凋亡和自噬相关蛋白的表达水平。结果 ALA-PDT对A431细胞的增殖活性有明显的抑制作用,且呈剂量和时间依赖性。与对照组比较,ALA-PDT组细胞增殖活性、线粒体膜电位均显著降低(P<0.05),而细胞凋亡与自噬水平显著提高(P<0.05),Cleaved-caspase-3、Bax、胞质Cyt C、LC-3Ⅱ、Beclin1等蛋白表达显著上调(P<0.05),Bcl-2表达显著下调(P<0.05)。与ALA-PDT组比较,ALA-PDT+3-MA组细胞增殖活性、线粒体膜电位以及细胞自噬水平显著降低(P<0.05),细胞凋亡水平显著提高(P<0.05),Cleaved-caspase-3、Bax、胞质Cyt C等蛋白表达显著上调(P<0.05),而Bcl-2、LC-3Ⅱ、Beclin1等蛋白表达显著下调(P<0.05)。结论 3-MA可通过抑制细胞自噬增强ALA-PDT对A431细胞产生的杀伤作用。
Objective To investigate the killing effect of 5-aminolevulinic acid-mediated photodynamic therapy(ALA-PDT) combined with 3-methyladenine(3-MA) on cutaneous squamous cell carcinoma cell line A431,and to explore its possible mechanism.Methods A431 cells were cultured in vitro and treated with ALA-PDT at different concentrations of ALA(0,0.20,0.40,0.80,1.60 mmol/L) for different time(12,24,48 h).Cell viability was detected by MTT assay and the value of IC50 was calculated.A431 cells were divided into four groups:control group,ALA-PDT group,3-MA group and ALA-PDT+3-MA group.After treatment,cell proliferation was detected by MTT method.Apoptosis rates(Annexin V-FITC/PI double staining) and the change of mitochondrial membrane potential(JC-1 staining) were detected by flow cytometry.Autophagy was observed by monodansylcadaverin(MDC) staining.The expression levels of apoptosis and autophagy related proteins were detected by Western blot.Results ALA-PDT significantly inhibited the proliferation activity of A431 cells in a dose-and time-dependent manner.Compared with control group,ALA-PDT significantly decreased the cell proliferation activity and mitochondrial membrane potential of A431 cells(P<0.05),elevated the levels of apoptosis and autophagy(P<0.05),increased the protein expression levels of Cleaved-caspase-3,Bax,LC-3Ⅱ,Beclin1 and cytoplasmic Cyt C(P<0.05),and decreased the protein expression of Bcl-2(P<0.05).Compared with the ALA-PDT group,the combination of 3-MA and ALA-PDT significantly decreased the cell proliferation activity,mitochondrial membrane potential and autophagy(P<0.05),elevated the level of apoptosis(P<0.05),increased the protein expression levels of Cleaved-caspase-3,Bax,cytoplasmic Cyt C(P<0.05),while decreased the protein expression levels of Bcl-2,LC-3Ⅱ and Beclin1(P<0.05).Conclusion 3-MA enhances the killing effect of ALA-PDT on A431 cells by inhibiting autophagy.
作者
王荣坤
林简
WANG Rongkun;LIN Jian(Dermatology&STD Department,Sanya People's Hospital,Sanya 572000,China)
出处
《中国皮肤性病学杂志》
CAS
CSCD
北大核心
2020年第6期627-633,共7页
The Chinese Journal of Dermatovenereology
