低剂量5-氨基酮戊酸光动力疗法对HaCaT细胞皮肤屏障相关蛋白表达的影响  

Effects of Low-dose Photodynamic Therapy with Aminolevulinic Acid on the Expression of Skin Barrier-Associated Proteins in HaCaT Cells

作　　者：王倩[1] 彭露 兰晓玲[1] 江雪梅[1] 张丽霞[1] 刘刚[1] 刘伟[1] WANG Qian;PENG Lu;LAN Xiaoling;JIANG Xuemei;ZHANG Lixia;LIU Gang;LIU Wei(The Institute of Dermatology and Venereology,Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital,Chengdu 610072,China;School of Medicine,University of Electronic Science and Technology of China,Chengdu 610054,China)

机构地区：[1]四川省医学科学院·四川省人民医院皮肤性病研究所,四川成都610072 [2]电子科技大学医学院,四川成都610054

出　　处：《中国皮肤性病学杂志》2023年第5期509-515,共7页The Chinese Journal of Dermatovenereology

基　　金：四川省科技厅重点研发计划项目(2022YFS0311);四川省卫健委科研项目(150222);四川省人民医院院科研基金项目(2016LY05)。

摘　　要：目的通过研究低剂量5-氨基酮戊酸(5-aminolevulinic acid,ALA)光动力疗法(photodynamic therapy,PDT)对HaCaT细胞中皮肤屏障相关蛋白表达的影响,探讨低剂量光动力改善皮肤屏障功能的潜在机制。方法以0、0.01、0.05、0.1、0.5及1 mmol/L ALA孵育HaCaT细胞后分别予以0、0.5、1.85、3.72及10 J/cm^(2)的(635±3)nm红光照射,CCK-8检测细胞存活率,摸索低剂量ALA-PDT促进HaCaT细胞活力的最佳ALA浓度及照光能量参数。将细胞分为对照组、光照组、ALA组、低剂量PDT组,分别予以相应处理后,用实时荧光定量PCR、Western blot实验检测细胞中丝聚合蛋白(filaggrin,FLG)、内皮蛋白(involucrin,IVL)、水通道蛋白3(aquaporin 3,AQP3)、紧密连接蛋白-1(claudin-1,CLDN1)、闭锁蛋白(occludin,OCLN)mRNA及蛋白表达。结果不同ALA浓度及不同照光能量处理HaCaT细胞的结果显示,在0.1 mmol/L ALA及3.72 J/cm^(2)照光能量的条件下,细胞的活力最佳。以该低剂量ALA-PDT处理HaCaT细胞后,FLG、IVL、AQP3、CLDN1及OCLN mRNA的相对表达量分别为10.25±6.34、5.10±2.74、4.89±1.33、8.53±6.43、4.06±2.01,对照组为1.01±0.01、1.02±0.02、1.01±0.01、1.01±0.00、1.02±0.01,光照组为2.64±1.04、2.29±1.49、2.49±1.87、4.51±4.41、1.57±1.12,ALA组为3.03±2.35、1.78±0.90、1.55±1.13、3.72±5.88、1.39±0.89,其中低剂量PDT组中FLG及AQP3表达明显升高,差异有统计学意义(P<0.05)。Wes-tern blot显示低剂量PDT组HaCaT细胞中FLG、IVL、AQP3、CLDN1蛋白表达水平均明显高于对照组、光照组及ALA组(P<0.05),OCLN的表达在几组中差异无统计学意义(P>0.05)。结论0.1 mmol/L ALA联合3.72 J/cm^(2)的照光能量为低剂量ALA-PDT对HaCaT细胞的最适ALA浓度及照光能量;低剂量ALA-PDT可能通过提高部分皮肤屏障相关蛋白的表达起到改善皮肤屏障的作用。Objective To explore the potential mechanism of low-dose 5-aminolevulinic acid(ALA)-photodynamictherapy(PDT)in improving skin barrier function by studying the effect of low-dose ALA-PDT on the expression of skin barrier-associated proteins in HaCaT cells.Methods HaCaT cells were incubated with ALA at the concentration of 0,0.01,0.05,0.1,0.5 and 1 mmol/L,and then irradiated with a red light source with an emission peak at(635±3)nm bandwidth.Total light dose to the cell culture positions were 0,0.5,1.85,3.72 and 10 J/cm^(2) respectively.Cell viability was determined using the cell counting kit(CCK)-8 and the optimal ALA concentration and light energy was explored for the viability of HaCaT cells.The cells were divided into 4 groups(control group,light group,ALA group and low-dose PDT group).After corresponding treatments,real-time fluorescent quantitative PCR and Western blot were conducted to determin the mRNA and protein expressions of filaggrin(FLG),involucrin(IVL),aquaporin 3(AQP3),claudin-1(CLDN1)and occludin(OCLN)in HaCaT cells.Results After treatments with different concentrations of ALA and irradiation at various energy densities,it was shown that 0.1 mmol/L ALA+3.72 J/cm^(2) phototreatment was the best parameter for low-dose ALA-PDT to promote cell viability in HaCaT cells.After treating HaCaT cells with this low-dose of ALA-PDT,the relative expression levels of FLG,IVL,AQP3,CLDN1 and OCLN mRNA were 10.25±6.34,5.10±2.74,4.89±1.33,8.53±6.43 and 4.06±2.01 respectively,the levels in the control group were 1.01±0.01,1.02±0.02,1.01±0.01,1.01±0.00,1.02±0.01,in light group were 2.64±1.04,2.29±1.49,2.49±1.87,4.51±4.41,1.57±1.12,and in ALA group were 3.03±2.35,1.78±0.90,1.55±1.13,3.72±5.88,1.39±0.89.The expression levels of FLG and AQP3 in the low-dose PDT group were significantly increased,and the difference was statistically significant(P<0.05).Western blot analysis showed that the FLG,IVL,AQP3 and CLDN1 protein expressions in the low-dose PDT group were significantly higher than those in con

关 键 词：光动力治疗 低剂量ALA-PDT 角质形成细胞 屏障蛋白 丝聚合蛋白 内皮蛋白 紧密连接蛋白质类 水通道蛋白3 

分 类 号：R751[医药卫生—皮肤病学与性病学]