摘要
目的 探讨过氧化氢（H2O2）对黑素细胞自噬的影响及可能的调节机制。方法 取对数生长期健康人黑素细胞，分为空白对照组（不予任何处理）、阳性对照组（100 nmol/L西罗莫司处理）和实验组（体积分数为10-7 - 10-3的H2O2处理），处理4 h后，使用CCK8法、流式细胞仪分别检测各组黑素细胞活性及凋亡率。吖啶橙染色检测自噬小泡，透射电镜下观察自噬小体，Western印迹检测自噬特异性蛋白Beclin 1、微管相关蛋白轻链3B（LC3B）。最后采用包含84个自噬相关基因的RT2 Profiler PCR Array筛选自噬相关的差异表达基因。结果 黑素细胞分别经10-3、5 × 10-4、10-4、5 × 10-5、10-5、5 × 10-6、10-6 H2O2处理后，细胞增殖活性及凋亡率与空白对照组相比差异均有统计学意义（F值分别为286.95、301.23，均P＜0.05），且随H2O2体积分数升高，增殖活性降低，凋亡率升高。除5 × 10-6 H2O2组分别与10-5、10-6 H2O2组间相比细胞凋亡率差异无统计学意义外，上述各H2O2组间两两比较，黑素细胞增殖活性及凋亡率差异均有统计学意义（P 〈 0.05）。吖啶橙染色及电镜观察发现，10-5 H2O2、10-6 H2O2和西罗莫司处理的黑素细胞中有自噬小体形成。Western印迹显示，10-5 H2O2、10-6 H2O2和西罗莫司组黑素细胞Beclin 1表达量和LC3B-Ⅱ/LC3B-Ⅰ比率均较空白对照组显著升高（P＜0.05）。RT2 Profiler PCR Array结果显示，与空白对照组相比，10-5 H2O2组、10-6 H2O2组和西罗莫司组中ATG12、ATG3、ULK1、PIK3CG、PTEN、PIK3C3表达均显著上调，EIF2AK3表达显著下调；10-5 H2O2组和西罗莫司组mTOR表达显著下调，ULK2表达显著上调；10-6 H2O2组mTOR表达未发生明显改变, AMPK、JNK1表达显著上调。结论 体积分数为10-5和10-6的H2O2均能有效诱导黑素细胞自噬，可能与影响相关信号分子表达相关。
Objective To evaluate the effect of hydrogen peroxide（H2O2） on autophagy in melanocytes, and to explore its possible regulatory mechanisms. Methods Normal human melanocytes at exponential growth phase were divided into several groups： blank control group receiving no treatment, positive control group treated with 100 nmol/L sirolimus solution, and experiment groups treated with H2O2 solution at different volume fractions of 10-7 - 10-3 respectively. After 4-hour treatment, cell counting kit-8 （CCK-8）assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively. Acridine orange staining was performed to detect autophagosome formation, transmission electron microscopy to observe ultrastructural changes of autophagosomes, and Western blot analysis to measure the of autophagy-specific protein Beclin 1 and microtubule-associated protein 1 light chain 3B （LC3B）. A total of 84 autophagy-related genes were analyzed by RT2 Profiler PCR Array, so as to screen differentially expressed autophagy-related genes. Results After the treatment with H2O2 at different volume fractions of 10-3, 5 × 10-4, 10-4, 5 × 10-5, 10-5, 5 × 10-6 and 10-6, experiment groups showed significantly decreased cellular proliferative activity, but significantly increased apoptosis rate compared with the blank control group （F = 286.95, 301.23, respectively, both P 〈 0.05）. With the increase in volume fractions of H2O2, the cellular proliferative activity was significantly gradually decreased （P 〈 0.05）, while the apoptosis rate showed an opposite trend （P 〈 0.05）, except that the 5 × 10-6 H2O2 group showed no significant differences in the apoptosis rate compared with the 10-5 H2O2 group and 10-6 H2O2 group. Acridine orange staining and electron microscopy showed autophagosome formation in the 10-5 H2O2 group, 10-6 H2O2 group and positive control group. Western blot analysis revealed that Beclin1 and LC3B-Ⅱ/LC3B-Ⅰ ratio were significantly
作者
龚晴丽
李雪
丁高中
凌雨婷
赵文娥
熊喜喜
鲁严
GongQingli Li Xue Ding Gaozhong Ling Yuting Zhao Wen＇e Xiong Xixi Lu Yah(Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Department of Analysis and Testing Center, Nanjing Medical University, Nanjing 210029, China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2017年第8期547-552,共6页
Chinese Journal of Dermatology
基金
国家自然科学基金（81171517）
江苏省“333工程”培养资金（BRA2013279）
关键词
黑素细胞
自噬
过氧化氢
氧化性应激
寡核苷酸序列分析
细胞凋亡
Melanocytes
Autophagy
Hydrogen peroxide
Oxidative stress
Oligonucleotide arraysequence analysis
Apoptosis
