摘要目的 探讨氨基酮戊酸光动力疗法(ALA-PDT)对成纤维细胞生长因子10(FGF-10)诱导的HaCaT细胞过度角化及增殖的作用及其机制.方法 将培养的HaCaT细胞分为4组:空白对照组(无任何处理),FGF-10组(加入FGF-10孵育24 h),ALA-PDT组(ALA孵育24 h后红光照射),FGF-10+ ALA-PDT组(FGF-10孵育24 h后,再加ALA孵育24 h,红光照射).用CCK-8法检测各组细胞增殖活性,Western印迹法检测K1、K6、K16及角质形成细胞生长因子受体(KGFR)的含量,ELISA法检测各组细胞上清液中白细胞介素1α(IL-1α)蛋白表达,免疫荧光检测各组细胞KGFR和K6蛋白表达水平.统计学分析采用析因设计的方差分析和单因素方差分析.结果 析因分析显示,ALA-PDT和FGF-10对HaCaT细胞增殖无交互作用(F交互=1.369,P=0.276),FGF-10对HaCaT细胞增殖有促进作用(FFGF-10=20.853,P＜0.05),而ALA-PDT有抑制作用(FALA-PDT=24.822,P＜ 0.05).与空白对照组细胞增殖活性(A值为0.924±0.024)比较,FGF-10组(1.233±0.099)显著升高(P＜0.05),而ALA-PDT组(0.718±0.107)显著降低(P＜0.05),FGF-10+ ALA-PDT组(0.901±0.014)较FGF-10组显著降低(P＜0.05).Western印迹法显示,FGF-10可促进K1、K6、K16蛋白和KGFR的表达(均P＜0.05),而ALA-PDT抑制这些蛋白的表达(均P＜ 0.05),FGF-10+ ALA-PDT组K1、K6、K16和KGFR的相对表达量与FGF-10组比较均显著降低(均P＜ 0.05).ELISA法显示,FGF-10会增加HaCaT细胞IL-1α蛋白分泌(P＜0.05),但ALA-PDT对其没有影响(P=0.467).此外,免疫荧光检测显示,FGF-10增加HaCaT细胞K6和KGFR免疫荧光强度(P＜0.05),而ALA-PDT降低K6和KGFR免疫荧光强度(P＜0.05),FGF-10+ ALA-PDT组K6和KGFR免疫荧光强度显著低于FGF-10组(P＜0.05).结论 ALA-PDT能抑制FGF-10诱导的HaCaT细胞过度角化及增殖,其机制与下调K1、K6、K16、KGFR及IL-1α有关.

abstractsObjective To evaluate the inhibitory effects of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on fibroblast growth factor 10 (FGF-10)-induced excessive cornification and proliferation of HaCaT cells,and to explore their mechanisms.Methods Cultured HaCaT cells were randomly divided into 4 groups:blank control group receiving no treatment,FGF-10 group treated with FGF-10 for 24 hours,ALA-PDT group pretreated with ALA for 24 hours followed by 635-nm red laser radiation,FGF-10 + ALA-PDT group pretreated with FGF-10 for 24 hours and then with ALA for another 24 hours followed by 635-nm red laser radiation.After additional culture for 24 hours,cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative activity of HaCaT cells,Western blot analysis to detect the protein expressions of keratin 1 (K1),K6,K16 and keratinocyte growth factor receptor (KGFR) in cells,enzyme-linked immunosorbent assay (ELISA) to measure the expression of interleukin 1α (IL-1α) in the culture supernatant of cells,and an immunofluorescence assay to analyze the protein expressions of KGFR and K6 in cells.Statistical analysis was carried out by using factorial analysis of variance (ANOVA) and one-way ANOVA.Results As factorial ANOVA showed,there was no interaction effect on the proliferation of HaCaT cells between ALA-PDT and FGF-10 (F =1.369,P =0.276),and main effect analysis revealed that the proliferation of HaCaT cells was accelerated by FGF-10 (F =20.853,P＜ 0.05),but inhibited by ALA-PDT (F=24.822,P＜ 0.05).The proliferative activity (expressed as the absorbence value at 490 nm) of HaCaT cells was significantly increased in the FGF-10 group (1.233 ± 0.099),but significantly decreased in the ALA-PDT group (0.718 ± 0.107) compared with the blank control group (0.924 ± 0.024,both P ＜ 0.05),and in the FGF-10 + ALA-PDT group (0.901 ± 0.014,P ＜ 0.05) compared with the FGF-10 group.Similarly,the protein expressions of K1,K6,K16,KGFR were significantly upregulated in the FG F-10 group,but significantly downregulated in the ALA-PDT group compared with the blank control group (all P ＜ 0.05),and in the FGF-10 + ALA-PDT group compared with the FGF-10 group (all P ＜ 0.05).The supernatant level of IL-1α was significantly elevated in the FGF-10 group (P ＜ 0.05),but showed no significant changes in the ALA-PDT group compared with the blank control group (P =0.467).Furthermore,the immunofluorescence intensities of K6 and KGFR in HaCaT cells were significantly enhanced in the FGF-10 group (both P＜0.05),but reduced in the ALA-PDT group compared with the blank control group (both P ＜ 0.05),and in the FGF-10 + ALA-PDT group compared with the FGF-10 group (both P＜ 0.05).Conclusion ALA-PDT can inhibit the FGF-10-induced excessive cornification and proliferation of HaCaT cells,likely by down-regulating K 1,K6,K16,KGFR and IL-1α.