中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (2): 86-90.

• 论著 • 上一篇    下一篇

喜树碱对HaCaT细胞自噬的影响

郝阳阳1,张梁宇2,王翔2,陆亚琪2,朱晓杨3,陈杨4   

  1. 1. 安徽医科大学解放军九八临床学院 解放军第九八医院皮肤科
    2. 浙江省湖州市解放军第九八医院
    3. 解放军98医院
    4. 浙江省湖州市解放军98医院
  • 收稿日期:2016-08-09 修回日期:2016-08-26 出版日期:2017-02-15 发布日期:2017-01-24
  • 通讯作者: 陈杨 E-mail:98cy@163.com
  • 基金资助:
    全军医学科技青年培育项目;南京军区医学科技创新重点项目

Effects of camptothecin on the autophagy of HaCaT cells

  • Received:2016-08-09 Revised:2016-08-26 Online:2017-02-15 Published:2017-01-24
  • Contact: Yang CHEN E-mail:98cy@163.com

摘要: 目的 探讨喜树碱对HaCaT细胞自噬的影响。方法 将HaCaT细胞分为对照组和实验组,对照组用0.1%二甲基亚砜处理,实验组分别用5、10、25、50、100、200 nmol/L浓度的喜树碱处理。不同浓度喜树碱作用于HaCaT细胞24、48 h,用CCK8法检测细胞增殖情况,流式细胞仪检测药物作用24 h后细胞凋亡情况,免疫印迹法检测自噬相关微管相关蛋白1轻链3(LC3)、p62的变化;选取10 nmol/L喜树碱作用于HaCaT细胞24 h,间接免疫荧光法检测细胞自噬蛋白LC3的变化。结果 5、10 nmol/L喜树碱对HaCaT细胞增殖、凋亡无显著影响,50、100、200 nmol/L喜树碱作用HaCaT细胞24 h,增殖抑制率分别为(31.23 ± 1.00)%,(54.21 ± 8.10)%,(66.75 ± 10.70)%;25、50、100、200 nmol/L喜树碱作用HaCaT细胞48 h,增殖抑制率分别为(25.81 ± 5.99)%、(44.35 ± 5.32)%、(65.81 ± 8.28)%、(73.23 ± 9.59)%,与同时段对照组相比,差异有统计学意义(均P < 0.001)。50、100、200 nmol/L喜树碱作用HaCaT细胞24 h,凋亡率分别为(14.46 ± 2.38)%、(19.15 ± 1.59)%、(29.88 ± 1.37)%,与对照组 (3.80 ± 0.13)%比较,差异有统计学意义(均P < 0.001)。5、10 nmol/L喜树碱处理HaCaT细胞24 h后,LC3Ⅱ表达上调,p62蛋白表达下调。间接免疫荧光显示10 nmol/L喜树碱作用HaCaT细胞24 h后,实验组与对照组自噬体阳性细胞百分率分别为(36.67 ± 4.55)%、(6.23 ± 0.92)%,两组差异有统计学意义(t = 6.546,P = 0.003)。结论 5、10 nmol/L喜树碱可诱导HaCaT细胞发生自噬,但对细胞增殖、凋亡无影响。50、100、200 nmol/L喜树碱抑制HaCaT细胞增殖,促使细胞发生凋亡,自噬水平降低。

Abstract: Hao Yangyang, Zhang Liangyu, Wang Xiang, Lu Yaqi, Zhu Xiaoyang, Chen Yang Department of Dermatology, 98th Hospital of People′s Liberation Army, 98th Clinical College of People′s Liberation Army, Anhui Medical University, Huzhou 313000, Zhejiang, China (Hao YY, Zhang LY, Lu YQ, Zhu XY, Chen Y); Department of Drug and Equipment, 98th Hospital of People′s Liberation Army, 98th Clinical College of People′s Liberation Army, Anhui Medical University, Huzhou 313000, Zhejiang, China (Wang X) Corresponding author: Chen Yang, Email: 98cy@163.com 【Abstract】 Objective To evaluate effects of camptothecin on the autophagy of HaCaT cells. Methods Some cultured HaCaT cells were divided into several groups to be treated with camptothecin at concentrations of 5, 10, 25, 50, 100 and 200 nmol/L, and 0.1% dimethyl sulfoxide (DMSO) (control group), respectively. Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferative activity of HaCaT cells after 24- and 48-hour treatment, flow cytometry to evaluate cell apoptosis after 24-hour treatment, and Western blot analysis to measure the of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and p62. Some HaCaT cells were divided into 2 groups to be treated with 10 nmol/L camptothecin and 0.1% DMSO for 24 hours, respectively. Then, indirect immunofluorescence assay (IFA) was performed to determine the LC3 . Results Camptothecin at low concentrations of 5 and 10 nmol/L had no significant effects on the proliferation and apoptosis of HaCaT cells. Compared with the control group, the cellular proliferative rates were significantly inhibited by (31.23 ± 1.00)%, (54.21 ± 8.10)% and (66.75 ± 10.70)% in the 50-, 100- and 200-nmol/L camptothecin groups after 24-hour treatment respectively, and by (25.81 ± 5.99)%, (44.35 ± 5.32)%, (65.81 ± 8.28)% and (73.23 ± 9.59)% in the 25-, 50-, 100- and 200-nmol/L camptothecin groups after 48-hour treatment respectively (all P < 0.001). After 24-hour treatment, the apoptosis rates were significantly higher in the 50-, 100- and 200-nmol/L camptothecin groups (14.46% ± 2.38%, 19.15% ± 1.59%, 29.88% ± 1.37%, respectively) than in the control group (3.80% ± 0.13%, all P < 0.001). After 24-hour treatment with 5 and 10 nmol/L camptothecin, the protein of LC3Ⅱ was significantly up-regulated, while p62 protein was significantly down-regulated. IFA showed that the percentage of autophagosome-positive cells was significantly higher in the 10-nmol/L camptothecin group than in the control group after 24-hour treatment (36.67% ± 4.55% vs. 6.23% ± 0.92%, t = 6.546, P = 0.003). Conclusions Camptothecin at low concentrations of 5 and 10 nmol/L can induce autophagy of HaCaT cells, but has no obvious effects on cell proliferation and apoptosis. Camptothecin at concentrations of 50, 100 and 200 nmol/L can inhibit cell proliferation, promote cell apoptosis, and decrease autophagy levels.