摘要目的 研究氨基酮戊酸光动力疗法(ALA-PDT)对皮肤鳞状细胞癌A431细胞蛋白激酶D1(PKD1)的影响,探讨ALA-PDT诱导A431细胞凋亡的机制.方法 体外培养A431细胞,用CCK-8法检测筛选出抑制作用最强的ALA浓度和光动力照射(PDT)剂量组合.将对数期生长A431细胞随机分为不予任何干预的对照组、ALA组、PDT组及ALA-PDT组,观察4组细胞接受相应干预12、24、36、48 h后细胞增殖抑制率和增殖抑制作用最强时间点的凋亡率.反转录PCR检测ALA-PDT作用A431细胞不同时间点后各组PKD1基因mRNA的表达.Western印迹法检测各组A431细胞中PKD1及其磷酸化位点Tyr463蛋白(pTyr463)、Ser916蛋白(pSer916)表达.结果 ALA浓度1.5 mmol/L、光照剂量2 J/cm2为最佳剂量组合.4组细胞干预12、24、36、48 h的增殖抑制率差异有统计学意义(F=39.56,P＜0.05).孵育24 h时:ALA-PDT组细胞增殖抑制率(46.26％±1.25％)高于ALA组(14.65％±0.33％)、PDT组(14.96％±0.68％)和对照组(11.98％±0.32％),差异有统计学意义(均P＜0.05);ALA-PDT组细胞增殖抑制率高于12 h(P＜ 0.05);4组细胞凋亡率差异有统计学意义(F=16.32,P＜0.05),ALA-PDT组(41.92％±3.23％)高于对照组(4.67％±0.88％)、ALA组(7.02％±1.52％)和PDT组(8.37％±0.59％),均P＜ 0.05.ALA-PDT作用于A431细胞后0、6、12、24、36、48 h,细胞PKD1基因mRNA的相对表达量差异有统计学意义(F=22.24,P＜0.05),孵育24 h mRNA相对表达量低于0、6、12h(均P＜0.05),与36、48 h差异无统计学意义(均P＞0.05).4组A431细胞中pSer916表达量差异无统计学意义(F=1.53,P＞0.05),PKD1和pTyr463表达量差异有统计学意义(F值为10.04、8.27,均P＜ 0.05),ALA-PDT组PKD1、pTyr463的表达低于对照组、ALA组、PDT组(均P＜0.05).结论 PKD1可能参与ALA-PDT疗法诱导A431细胞凋亡的光化学反应进程,且可能是通过Tyr463位点活化促进癌变的发展.

abstractsObjective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431,and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 ceils.Methods A431 cells were cultured in vitro,and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect.A431 ceils at exponential growth phase were randomly divided into 4 groups:control group receiving no treatment,ALA group treated with ALA solution alone,PDT group treated with PDT alone,and ALA-PDT group treated firstly with ALA solution and then with PDT.After 12-,24-,36-and 48-hour additional culture,CCK-8 assay was conducted to evaluate the cellular proliferation inhibition,and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry.RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment,and Western blot analysis to measure protein expression of PKD 1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells.Results The combination of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect.After 12-,24-,36-and 48-hour additional culture,there were significant differences in the proliferation inhibition rate among the 4 groups (F =39.56,P ＜ 0.05).At 24 hours after the treatment,the ALA-PDT group showed significantly higher proliferation inhibition rate (46.26％ ± 1.25％) compared with the ALA group (14.65％ ± 0.33％,P ＜ 0.05),PDT group (14.96％ ± 0.68％,P ＜ 0.05) and control group (11.98％ ± 0.32％,P ＜ 0.05),as well as compared with that at 12 hours (P ＜ 0.05).At 24 hours after the treatment,the apoptosis rate significantly differed among the 4 groups (F =16.32,P ＜ 0.05),and the ALA-PDT group showed a significantly higher apoptosis rate (41.92％ ± 3.23％) compared with the control group (4.67％ ± 0.88％,P ＜ 0.05),ALA group (7.02％ ± 1.52％,P ＜ 0.05) and PDT group (8.37％ ± 0.59％,P ＜ 0.05).At 0,6,12,24,36 and 48 hours after the treatment,there were significant differences in the mRNA expression of PRKD 1 among the 4 groups (F =22.24,P ＜ 0.05),and the mRNA expression of PRKD1 at 24 hours was significantly lower than that at 0,6,12 hours (all P ＜ 0.05),but was not significantly different from that at 36 and 48 hours (both P ＞ 0.05).No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F =1.53,P ＞ 0.05),while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD 1 among the 4 groups (F =10.04,8.27,both P ＜ 0.05).Additionally,the ALA-PDT group showed significantly lower expression of PKD 1 and Tyr463-phosphorylated PKD 1 compared with the control group,ALA group and PDT group (all P ＜ 0.05).Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT,and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation.