中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (6): 383-388.doi: 10.3760/cma.j.issn.0412-4030.2019.06.003

• 论著 • 上一篇    下一篇

体外低浓度过氧化氢对人黑素细胞自噬水平的影响及自噬相关lncRNA的筛选

石家绮1    李雪2    孙利1    赵文娥3    丁书红1    侯晓媛1    修艳燕1    鲁严1   

  1. 1南京医科大学第一附属医院皮肤科  210029;2南京医科大学附属妇产医院皮肤科  210004;3南京医科大学分析测试中心电镜室  210000
  • 收稿日期:2019-01-02 修回日期:2019-04-10 发布日期:2019-06-03
  • 通讯作者: 鲁严 E-mail:luyan6289@163.com
  • 基金资助:
    国家自然科学基金(81171517、81872541、81602774)

In vitro effect of low-concentration hydrogen peroxide on autophagy in human melanocytes and screening for autophagy-related lncRNAs

Shi Jiaqi1, Li Xue2, Sun Li1, Zhao Wen′e3, Ding Shuhong1, Hou Xiaoyuan1, Xiu Yanyan1, Lu Yan1   

  1. 1Department of Dermatology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; 2Department of Dermatology, Women′s Hospital of Nanjing Medical University, Nanjing 210004, China; 3Laboratory of Electron Microscopy, Department of Analysis and Testing Center, School of Basic Medical Science, Nanjing Medical University, Nanjing 210000, China
  • Received:2019-01-02 Revised:2019-04-10 Published:2019-06-03
  • Contact: Lu Yan E-mail:luyan6289@163.com
  • Supported by:
    National Natural Science Foundation of China (81171517, 8187254, 81602774)

摘要: 【摘要】 目的 研究低浓度过氧化氢(H2O2)处理对人黑素细胞黏附功能及自噬水平的影响,并筛选自噬相关lncRNA。方法 从健康男性包皮环切术后获得的皮肤组织中分离、培养黑素细胞。对数生长期黑素细胞分为对照组(无H2O2处理)、H2O2组(400 μmol/L H2O2处理)和H2O2 + NAC组(4 mmol/L NAC预处理细胞2 h,然后加入400 μmol/L H2O2)。处理5 d后,用免疫荧光检测上述各组黑素细胞E?钙黏素、微管相关蛋白1轻链3(LC3)及p62表达,透射电镜观察细胞结构及自噬体,Western印迹法检测自噬相关蛋白LC3和p62的表达,运用基因芯片技术筛选自噬相关lncRNA。采用Graphpad Prism 6软件进行统计分析,多组间比较采用单因素方差分析,并用Tukey检验进行两两间的多重比较。结果 共聚焦显微镜下,与对照组和H2O2 + NAC组相比,H2O2组黑素细胞E?钙黏素及LC3荧光强度均明显下降,而p62荧光强度增高,且电镜下H2O2组黑素细胞内自噬体数量减少。Western印迹结果显示,H2O2处理组黑素细胞LC3?Ⅱ/LC3?Ⅰ值(0.604 ± 0.012)显著低于对照组(1.200 ± 0.081,q = 7.718,P < 0.01)及H2O2 + NAC组(1.017 ± 0.062,q = 5.076,P < 0.05),同时其p62/β肌动蛋白值(0.881 ± 0.079)显著高于对照组(0.456 ± 0.121,q = 4.847,P < 0.05)及H2O2 + NAC组(0.492 ± 0.049,q = 4.439,P < 0.05)。H2O2 + NAC组与对照组的LC3?Ⅱ/LC3?Ⅰ值及p62/β肌动蛋白值差异均无统计学意义(P > 0.05)。基因芯片结果显示,与对照组相比,H2O2组中有18个自噬相关lncRNA与黑素细胞提前衰老有关,从中筛选出了明显上调的自噬相关lncRNA NONHSAT190308.1(差异 > 10倍)。结论 低浓度H2O2可使黑素细胞E-钙黏素表达下降,降低黑素细胞自噬水平,上调自噬相关lncRNA NONHSAT190308.1表达水平。

关键词: 白癜风, 黑素细胞, 细胞黏附, 自噬, 氧化性应激, 过氧化氢, 微管相关蛋白质类, E-钙黏素, 长链非编码RNA

Abstract: 【Abstract】    Objective    To evaluate the effect of the treatment with low-concentration hydrogen peroxide (H2O2) on the adhesive function of and autophagy in human melanocytes, and to screen long noncoding RNAs (lncRNAs) related to autophagy. Methods    Melanocytes were isolated from foreskins of healthy males after circumcision, and subjected to cultivation. Melanocytes at exponential growth phase were divided into 3 groups: control group receiving no treatment, H2O2 group treated with 400 μmol/L H2O2, and H2O2 + NAC group pretreated with 4 mmol/L NAC for 2 hours followed by the treatment with 400 μmol/L H2O2. After 5-day treatment, immunofluorescence study was performed to determine the expression of E-cadherin, microtubule-associated protein 1 light chain 3 (LC3)and p62, and Western blot analysis to determine the expression of autophagy-related protein LC3 and p62. Cell structures and autophagosomes were observed by transmission electron microscopy, and autophagy-related lncRNAs were screened using gene chip technology. Statistical analysis was done with Graphpad Prism 6 software using one-way analysis of variance for comparison among groups, and Tukey′s test for multiple comparisons. Results    Under the confocal microscopy, the H2O2 group showed significantly decreased fluorescence intensity of E-cadherin and LC3 in the melanocytes and decreased number of autophagosomes in melanocytes, but significantly increased fluorescence intensity of p62 compared with the control group and H2O2 + NAC group. Western blot analysis showed that the LC3-Ⅱ/LC3-Ⅰratio in the melanocytes was significantly lower in the H2O2 group (0.604 ± 0.012) than in the control group (1.200 ± 0.081, q = 7.718, P < 0.01) and H2O2 + NAC group (1.017 ± 0.062, q = 5.076, P < 0.05), while the p62/β-actin ratio in the melanocytes was significantly higher in the H2O2 group (0.881 ± 0.079) than in the control group (0.456 ± 0.121, q = 4.847, P < 0.05) and H2O2 + NAC group (0.492 ± 0.049, q = 4.439, P < 0.05). There were no significant differences in the LC3-Ⅱ/LC3-Ⅰratio or p62/β-actin ratio between the H2O2 + NAC group and control group (P > 0.05). Gene chip technology showed that 18 autophagy-related lncRNAs were associated with premature senescence of melanocytes and differentially expressed in the H2O2 group compared with the control group, and the autophagy-related lncRNA NONHSAT190308.1(> 10-fold increase) was screened out. Conclusion    Low-concentration H2O2 can decrease the expression of E-cadherin and the level of autophagy in melanocytes, and can up-regulate the expression of autophagy-associated lncRNA NONHSAT190308.1. 

Key words: Vitiligo, Melanocytes, Cell adhesion, Autophagy, Oxidative stress, Hydrogen peroxide, Microtubule-associated proteins, E-cadherin, LncRNA

中图分类号: 

  • R758.4+1

引用本文

石家绮 李雪 孙利 赵文娥 丁书红 侯晓媛 修艳燕 鲁严. 体外低浓度过氧化氢对人黑素细胞自噬水平的影响及自噬相关lncRNA的筛选[J]. 中华皮肤科杂志, 2019,52(6):383-388. doi:10.3760/cma.j.issn.0412-4030.2019.06.003

Shi Jiaqi, Li Xue, Sun Li, Zhao Wen′e, Ding Shuhong, Hou Xiaoyuan, Xiu Yanyan, Lu Yan. In vitro effect of low-concentration hydrogen peroxide on autophagy in human melanocytes and screening for autophagy-related lncRNAs[J]. Chinese Journal of Dermatology, 2019, 52(6): 383-388.doi:10.3760/cma.j.issn.0412-4030.2019.06.003