喜树碱对人原代角质形成细胞自噬的影响

目的 探讨喜树碱对人原代角质形成细胞（HPK）自噬的影响。方法 取健康男性包皮组织，采用两步消化法分离提取HPK，取第3代细胞用于实验。将HPK分为实验组和对照组，实验组用200 nmol/L、2 μmol/L、6 μmol/L喜树碱处理，对照组用0.1%二甲基亚砜（DMSO）处理。处理24、48 h后，采用CCK8法检测细胞增殖；流式细胞仪检测药物作用24 h后细胞凋亡；免疫印迹法检测微管相关蛋白1轻链3（LC3）、p62变化。选取2 μmol/L喜树碱作用HPK 24 h，间接免疫荧光法检测LC3蛋白变化，透射电镜观察自噬体超微结构，进一步确认喜树碱对自噬的诱导效应。结果 随着喜树碱浓度的增加，对HPK的增殖抑制作用逐渐增强，各组24 h增殖抑制率总体差异有统计学意义（F = 152.9，P < 0.01），其中2、6 μmol/L组高于对照组，差异有统计学意义（t = 12.09、18.76，均P < 0.01），200 nmol/L组与对照组差异无统计学意义（t = 2.24，P > 0.05）。48 h时各组增殖抑制率总体差异有统计学意义（F = 123.8，P < 0.01），实验组均高于对照组（均P < 0.01）。对照组和200 nmol/L、2 μmol/L、6 μmol/L喜树碱组24 h细胞凋亡率分别为（2.30 ± 1.68）%、（15.90 ± 2.14）%、（29.33 ± 3.51）%、（35.28 ± 3.05）%，差异有统计学意义（F = 89.57，P < 0.01），各实验组与对照组比较差异均有统计学意义（均P < 0.01）。2、6 μmol/L喜树碱处理细胞24 h后，LC3Ⅱ蛋白显著上调，p62蛋白表达下调。间接免疫荧光检测显示，2 μmol/L喜树碱组与对照组自噬体阳性细胞比例分别为（60.16 ± 8.78）%、（38.96 ± 13.12）%，差异有统计学意义（t = 3.003，P < 0.05）。透射电镜观察到2 μmol/L喜树碱作用细胞24 h后细胞内形成自噬体和自噬溶酶体。结论 2、6 μmol/L喜树碱可诱导HPK自噬水平升高，同时抑制细胞增殖、诱导细胞凋亡。

Effects of camptothecin on autophagy of human primary keratinocytes

Hao Yangyang, Zhang Liangyu, Wang Xiang, Tong Yunfeng, Sun Ying, Chen YangDepartment of Dermatology, 98th Hospital of People′s Liberation Army, Huzhou 313000, Zhejiang, China（Hao YY ［current affiliation: Department of Dermatology, The First People′s Hospital of Huzhou, Huzhou 313000, Zhejiang, China］, Zhang LY, Tong YF, Sun Y, Chen Y）; Department of Drug and Equipment, 98th Hospital of People′s Liberation Army, Huzhou 313000, Zhejiang, China （Wang X）Corresponding author: Chen Yang, Email: 98cy@163.com【Abstract】 Objective To evaluate the effect of camptothecin on the autophagy of human primary keratinocytes （HPKs）. Methods HPKs were isolated from foreskin tissues of healthy males by a two-step digestion method, and the third-passage cells were used for following experiments. These HPKs were randomly divided into several groups: experimental groups treated with camptothecin at concentrations of 200 nmol/L, 2 and 6 μmol/L separately, and a control group treated with 0.1% dimethyl sulfoxide （DMSO）. After 24- and 48-hour treatment, cell counting kit-8 （CCK-8）assay was conducted to estimate the proliferative activity of HPKs. Flow cytometry was performed to detect cell apoptosis after 24-hour treatment, and Western blot analysis to measure the of autophagy-associated proteins such as microtubule-associated protein 1 light chain-3 （LC3） and p62. Some other HPKs were treated with 2 μmol/L camptothecin for 24 hours. Indirect immunofluorescence assay was performed to observe changes in LC3 , and transmission electron microscopy to observe the ultrastructure of autophagosomes, so as to further validate the inductive effect of camptothecin on autophagy. Results The inhibitory effect of camptothecin on the proliferation of HPKs gradually increased along with the increase of camptothecin concentration, and there was a significant difference in the proliferation inhibition rates among the experimental groups and control group at 24 hours （F = 152.9, P < 0.01）. Additionally, the proliferation inhibition rates were significantly higher in the 2-, 6-μmol/L camptothecin groups than in the control group （t = 12.09, 18.76, both P < 0.01）, but there was no significantly difference between the 200-μmol/L camptothecin group and control group （t = 2.24, P > 0.05）. At 48 hours, there was still a significant difference in the proliferation inhibition rates among the experimental groups and control group （F = 123.8, P < 0.01）, and all the experimental groups showed increased proliferation inhibition rates compared with the control group （all P < 0.01）. At 24 hours, the cell apoptosis rates also significantly differed among the control group, 200-nmol/L, 2-μmol/L and 6-μmol/L camptothecin groups （2.30% ± 1.68%, 15.90% ± 2.14%, 29.33% ± 3.51%, 35.28% ± 3.05%, respectively; F = 89.57, P < 0.01）, and all the three experimental groups showed higher cell apoptosis rates compared with the control group （all P < 0.01）. After 24-hour treatment with 2 or 6 μmol/L camptothecin, the protein of LC3Ⅱ were significantly up-regulated, but the protein of p62 was significantly down-regulated. Indirect immunofluorescence assay showed that the percentage of autophagosome-positive cells was significantly higher in the 2-μmol/L camptothecin group than in the control group （60.16% ± 8.78% vs. 38.96% ± 13.12%, t = 3.003, P < 0.05）. After 24-hour treatment with 2 μmol/L camptothecin, autophagosomes and autolysosomes were observed in HPKs with a transmission electron microscope. Conclusion Camptothecin at concentrations of 2 and 6 μmol/L can increase the autophagy level in HPKs, meanwhile, inhibit cell proliferation and induce cell apoptosis.