氨基酮戊酸光动力疗法对鳞状细胞癌细胞株A431蛋白激酶D1及其磷酸化位点的影响  

Effect of aminolevulinic acid-based photodynamic therapy on the expression of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell Hne A431

作　　者：顾静 王付亮 王来群[1] 刘保国[3] 周萌 苗国英[3] 李小静[3] 

机构地区：[1]河南宏力医院皮肤科,河南长垣453400 [2]河北医科大学研究生学院 [3]河北工程大学附属医院皮肤科

出　　处：《中华皮肤科杂志》2018年第2期96-100,共5页Chinese Journal of Dermatology

基　　金：河北省2016年度科技支撑计划项目（16277724D）;河北省政府资助临床医学优秀人才培养项目（361037）

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、12 h（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Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy （ALA-PDT） on the 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 cells. 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 cells 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 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 of PKD1 and its phosphorylation at Tyr463 （pTyr463） and Ser916 （pSer916） in A431 cells. Results The combi-nation 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 signif

关 键 词：光化学疗法 氨基酮戊酸 肿瘤 鳞状细胞 蛋白激酶类 原代细胞培养 A431细胞 

分 类 号：R739.5[医药卫生—肿瘤]