中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (12): 899-903.doi: 10.3760/cma.j.issn.0412-4030.2017.12.009

• 论著 • 上一篇    下一篇

H2O2对人皮肤成纤维细胞衰老标记蛋白30及自噬相关蛋白LC3Ⅱ表达的影响

田黎明    彭圆    胡荣毅    程杨    姜红浩    陈红英    田庆均    张翀    王平   

  1. 430022 武汉，湖北中医药大学附属中西医结合医院  华中科技大学附属武汉中西医结合医院  武汉市第一医院皮肤科（田黎明、胡荣毅、程杨、姜红浩、陈红英、田庆均）；湖北中医药大学基础医学院（彭圆、张翀、王平）
  • 收稿日期:2016-12-19 修回日期:2017-08-04 出版日期:2017-12-15 发布日期:2017-11-30
  • 通讯作者: 王平 E-mail:pwang@aliyun.com
  • 基金资助:
    国家自然科学基金;国家自然科学基金;国家自然科学基金;中国博士后科学基金;湖北省自然科学基金;湖北省自然科学基金;湖北省卫生计生委科学基金;武汉市卫生计生委科学基金;武汉市卫生计生委科学基金;武汉市中青年医学骨干人才培养工程基金

Effect of hydrogen peroxide on senescence marker protein-30 and autophagy-related protein LC3-Ⅱ in human skin fibroblasts

Tian Liming, Peng Yuan, Hu Rongyi, Cheng Yang, Jiang Honghao, Chen Hongying, Tian Qingjun, Zhang Chong, Wang Ping      

  1. Department of Dermatology, Wuhan No.1 Hospital, Hospital of Traditional Chinese and Western Medicine Affiliated to Hubei University of Chinese Medicine, Wuhan Hospital of Traditional Chinese and Western Medicine Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China（Tian LM, Hu RY, Cheng Y, Jiang HH, Chen HY, Tian QJ）; College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065, China （Peng Y, Zhang C, Wang P）
  • Received:2016-12-19 Revised:2017-08-04 Online:2017-12-15 Published:2017-11-30
  • Supported by:
    National Natural Science Foundation of China;National Natural Science Foundation of China;National Natural Science Foundation of China;China Postdoctoral Science Foundation;Natural Science Foundation of Hubei Province of China;Natural Science Foundation of Hubei Province of China;Science Foundation of Health and Family Planning Commission of Hubei Province;Science Foundation of Health and Family Planning Commission of Wuhan Municipality;Science Foundation of Health and Family Planning Commission of Wuhan Municipality;Training Project for Young and Middle-aged Core Talents of Health System of Wuhan City

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摘要： 目的 探讨H2O2对人皮肤成纤维细胞（NHSF）衰老标记蛋白30（SMP30）以及细胞自噬相关蛋白微管相关蛋白1轻链3Ⅱ型（LC3Ⅱ）的影响。方法 用150 μmol/L H2O2处理2 ~ 4代NHSF 2 h，构建细胞衰老模型作为H2O2组，而未处理的NHSF作为对照组。细胞衰老β半乳糖苷酶（SA?β?gal）染色法检测衰老细胞比例，间接免疫荧光检测自噬相关蛋白LC3的表达，反转录PCR（RT?PCR）检测SMP30 mRNA的表达，Western印迹法检测SMP30和LC3蛋白的表达。结果 H2O2组NHSF的SA?β?gal染色阳性率（41.70% ± 2.95%）显著高于对照组（3.03% ± 0.25%，t = 22.59，P < 0.05）。间接免疫荧光结果显示，H2O2组NHSF LC3阳性率（12.60% ± 1.57%）明显低于对照组（23.67% ± 3.04%，t = 5.61，P < 0.05）。Western印迹显示，H2O2组LC3Ⅰ/GAPDH比值（0.40 ± 0.02）与对照组（0.41 ± 0.04）比较差异无统计学意义（P > 0.05），而H2O2组LC3Ⅱ/GAPDH比值（0.20 ± 0.02）明显低于对照组（0.80 ± 0.03，t = 29.69，P < 0.05），且H2O2组LC3Ⅱ/LC3Ⅰ比值（0.51 ± 0.03）亦明显低于对照组（1.98 ± 0.23，t = 10.967，P < 0.05）。H2O2组SMP30 mRNA和蛋白水平（与GAPDH mRNA和蛋白的比值分别为0.16 ± 0.01和0.27 ± 0.02）明显低于对照组（分别为0.35 ± 0.01和0.63 ± 0.02），均P < 0.05。结论 H2O2可降低NHSF中SMP30及LC3Ⅱ的表达水平，加速NHSF衰老。

Abstract: Tian Liming, Peng Yuan, Hu Rongyi, Cheng Yang, Jiang Honghao, Chen Hongying, Tian Qingjun, Zhang Chong, Wang Ping Department of Dermatology, Wuhan No.1 Hospital, Hospital of Traditional Chinese and Western Medicine Affiliated to Hubei University of Chinese Medicine, Wuhan Hospital of Traditional Chinese and Western Medicine Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China（Tian LM, Hu RY, Cheng Y, Jiang HH, Chen HY, Tian QJ）; College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065, China （Peng Y, Zhang C, Wang P） Corresponding author: Wang Ping, Email: pwang@aliyun.com 【Abstract】 Objective To evaluate the effect of hydrogen peroxide （H2O2） on a senescence marker protein-30 （SMP30） and an autophagy-related protein microtubule-associated protein 1 light chain 3 typeⅡ （LC3-Ⅱ） in normal human skin fibroblasts （NHSFs）. Methods NHSFs were isolated from the foreskin of children, and subjected to culture in vitro. The second- to fourth-passage NHSFs were treated with 150 μmol/L H2O2 for 2 hours to establish a model for cellular senescence, while un-treated NHSFs served as control group. Senescence-associated β-galactosidase （SA-β-gal） staining was performed to determine the percentage of senescent cells, indirect immunofluorescence assay to determine the of the autophagy-related protein LC3, reverse transcription PCR （RT-PCR） to measure the mRNA of SMP30, and Western blot analysis to measure the protein of SMP30 and LC3. Results The percentage of senescent （SA-β-gal-positive） cells was significantly higher in the H2O2 group than in the control group （41.70% ± 2.95% vs. 3.03% ± 0.25%, t = 22.59, P < 0.05）. Indirect immunofluorescence assay showed that the percentage of LC3-positive cells was significantly lower in the H2O2 group than in the control group （12.60% ± 1.57% vs. 23.67% ± 3.04%, t = 5.61, P < 0.05）. As Western blot analysis showed, no significant difference in the of LC3-Ⅰ（LC3-Ⅰ/glyceraldehyde-3-phosphate dehydrogenase ［GAPDH］ ratio） was observed between the H2O2 group and control group （0.40 ± 0.02 vs. 0.41 ± 0.04, P > 0.05）, while the H2O2 group showed significantly lower of LC3-Ⅱ（LC3-Ⅱ/GAPDH ratio: 0.20 ± 0.02 vs. 0.80 ± 0.03, t = 29.69, P < 0.05） and lower LC3-Ⅱ/LC3-Ⅰratio （0.51 ± 0.03 vs. 1.98 ± 0.23, t = 10.967, P < 0.05） compared with the control group. Moreover, the mRNA and protein of SMP30 （SMP30/GAPDH ratio） was significantly lower in the H2O2 group than in the control group （mRNA: 0.16 ± 0.01 vs. 0.35 ± 0.01; protein: 0.27 ± 0.02 vs. 0.63 ± 0.02, both P < 0.05）. Conclusion H2O2 can decrease the of SMP30 and LC3-Ⅱ in NHSFs, and accelerate the senescence of NHSFs.