中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (2): 96-100.doi: 10.3760/cma.j.issn.0412-4030.2018.02.003

• 论著 • 上一篇    下一篇

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

顾静1,王付亮2,王来群1,刘保国3,周萌4,苗国英5,李小静6   

  1. 1. 河南宏力医院
    2. 河北医科大学研究生学院
    3. 河北工程大学附属医院
    4. 承德医学院 研究生学院
    5. 河北省邯郸市河北工程大学附属医院皮肤科
    6. 河北邯郸 河北工程大学附属医院
  • 收稿日期:2017-01-17 修回日期:2017-06-21 发布日期:2018-01-30
  • 通讯作者: 刘保国 E-mail:lbg66@163.com
  • 基金资助:

    河北省2016年度科技支撑计划项目;河北省政府资助临床医学优秀人才培养项目;河北省科技计划项目

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

  • Received:2017-01-17 Revised:2017-06-21 Published:2018-01-30
  • Supported by:

    Science Supply Plan of Hebei Province in 2016;Talents Training Program in Clinical Medicine Sponsored by Hebei Government

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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进程，且可能是通过Tyr463位点活化促进癌变的发展。

Abstract:

Gu Jing, Wang Fuliang, Wang Laiqun, Liu Baoguo, Zhou Meng, Miao Guoying, Li Xiaojing Department of Dermatology, Henan Hongli Hospital, Changyuan 453400, Henan, China （Gu J, Wang LQ）; Graduate School of Hebei Medical University, Shijiazhuang 05000, China （Wang FL）; Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan 056002, Hebei, China（Liu BG, Zhou M, Miao GY, Li XJ） Corresponding author: Liu Baoguo, Email: lbg66@163.com 【Abstract】 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 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 of PRKD1 among the 4 groups （F = 22.24, P < 0.05）, and the mRNA 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 was found among the 4 groups （F = 1.53, P > 0.05）, while there were significant differences in the of PKD1 and Tyr463-phosphorylated PKD1 among the 4 groups （F = 10.04, 8.27, both P < 0.05）. Additionally, the ALA-PDT group showed significantly lower of PKD1 and Tyr463-phosphorylated PKD1 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.

引用本文

顾静 王付亮 王来群 刘保国 周萌 苗国英 李小静. 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[J]. 中华皮肤科杂志, 2018,51(2):96-100. doi:10.3760/cma.j.issn.0412-4030.2018.02.003