中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (3): 182-185.doi: 10.3760/cma.j.issn.0412-4030.2018.03.004

• 论著 • 上一篇    下一篇

定量检测绿色荧光蛋白-轻链蛋白3转基因小鼠角质形成细胞自噬水平

金婷婷1,Heidemarie Rossiter2,Michael Mildner2,Florian Gruber2,Leopold Eckhart3,Erwin Tschachler4,赵邑5   

  1. 1. 北京大学第一医院
    2.
    3. Department of Dermatology, Medical University of Vienna
    4. Department of Dermatology, Medical University of Vienna, Vienna, Austria
    5. 北京清华长庚医院
  • 收稿日期:2017-01-13 修回日期:2017-11-06 出版日期:2018-03-15 发布日期:2018-03-06
  • 通讯作者: 赵邑 E-mail:zhaoyi@btch.edu.cn
  • 基金资助:
    国家自然科学基金;国家自然科学基金

Quantitative analysis of autophagy in green fluorescent protein-light chain 3 transgenic murine keratinocytes

Ting-ting JIN1, 2, 3, 3, 3,Erwin Tschachler4,Yi 0ZHAO   

  • Received:2017-01-13 Revised:2017-11-06 Online:2018-03-15 Published:2018-03-06
  • Contact: Yi 0ZHAO E-mail:zhaoyi@btch.edu.cn
  • Supported by:
    National Natural Science Foundation of China;National Natural Science Foundation of China

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摘要： 目的 探索一种高通量定量检测细胞自噬小体数量的方法。方法 将绿色荧光蛋白?轻链蛋白3（GFP?LC3）转基因小鼠的角质形成细胞分为对照组（不接受照射或饥饿处理）、饥饿组（给予饥饿处理）、20 J/cm2长波紫外线（UVA）照射组和40 J/cm2 UVA照射组。处理后6 h，将细胞固定后在共聚焦显微镜下采集图片。用ImageJ软件编辑宏指令对细胞内GFP?LC3绿色荧光点及细胞进行自动计数，并比较不同组间自噬水平。结果 利用ImageJ软件编辑的宏指令可以成功识别并计数荧光图片中的自噬小体。在测试计算机上分析一张420万像素的图片仅需不到0.6 s。饥饿组、20 J/cm2 UVA照射组和40 J/cm2 UVA照射组平均自噬小体数量高于对照组，未加胃抑素A时各组间比较，F = 5.01，P < 0.05；加入胃抑素A时各组间比较，F = 20.05，P < 0.05。该方法可以成功区分饥饿组、UVA照射组和对照组的自噬水平差异。结论 成功为自噬研究提供了一种新的高通量定量检测方法，该方法能够快速准确地检测细胞自噬荧光点。

关键词: 自噬, 角蛋白细胞, 绿色荧光蛋白质类, 显微镜检查, 共焦, 图像处理, 计算机辅助

Abstract: Jin Tingting, Heidemarie Rossiter, Michael Mildner, Florian Gruber, Leopold Eckhart, Erwin Tschachler, Zhao Yi Department of Dermatology and Venereology, Peking University First Hospital, Beijing 100034, China（Jin TT ［the current affiliation: Plastic and Reconstructive Surgery, Zhejiang Provincial People′s Hospital, Hangzhou 310014, China］）; Research Department of Biology and Pathobiology of the Skin, Medical University of Vienna, Vienna 1090, Austria （Rossiter H, Mildner M, Gruber F, Eckhart L, Tschachler E）; Department of Dermatology, Beijing Tsinghua Changgung Hospital, Affiliated to Tsinghua University, Beijing 102218, China （Zhao Y） Corresponding authors: Erwin Tschachler, Email: erwin.tschachler@meduniwien.ac.at; Zhao Yi, Email: zhaoyi@btch.edu.cn 【Abstract】 Objective To explore a high-throughput method for quantitative analysis of autophago-somes. Methods Green fluorescent protein-light chain 3（GFP-LC3）transgenic murine keratinocytes were randomly divided into 4 groups: control group receiving no treatment, starvation group subjected to starved culture, 20 J/cm2 ultraviolet A （UVA） group treated with 20 J/cm2 UVA radiation, and 40 J/cm2 UVA group treated with 40 J/cm2 UVA radiation. After 6-hour treatment, the cells were fixed, and images were acquired by confocal laser scanning microscopy. A macro was created by the ImageJ software to automatically quantify the GFP-LC3 puncta in the cells and the number of cells. Then, the level of autophagy was compared among different groups. Results By using the macro created by the ImageJ software, autophago-somes in the keratinocytes were successfully identified and quantified. Less than 0.6 second was needed for analyzing an image of 4.2 mega pixels in a test computer. The average number of autophagosomes in keratinocytes was significantly higher in the starvation group, 20-J/cm2 UVA group and 40-J/cm2 UVA group than in the control group whether with the treatment with pepstatin A （F = 20.05, P < 0.05） or not （F = 5.01, P < 0.05）. This method could successfully differentiate the autophagy levels among the starvation group, UVA irradiation groups and control group. Conclusion A new high-throughput method, which can rapidly and accurately quantify GFP-LC3 puncta in cells, is established successfully to quantificationally detect autophagy.

Key words: Autophagy, Keratinocytes, Green fluorescent proteins, Microscopy, confocal, Image processing, computer-assisted

中图分类号: 

  • R751