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山东大学学报 (医学版) ›› 2021, Vol. 59 ›› Issue (4): 17-27.doi: 10.6040/j.issn.1671-7554.0.2021.0288

• 基础医学 • 上一篇    下一篇

氧糖剥夺条件下培养表皮干细胞的定量蛋白质组学分析

张华宇,殷思源,刘健,马嘉旭,宋茹,曹国起,王一兵   

  1. 山东大学, 山东省千佛山医院整形外科学, 济南市组织工程皮肤再生与创面修复临床医学研究中心, 山东 济南 250014
  • 发布日期:2021-04-30
  • 通讯作者: 王一兵. E-mail:ybwang@sdfmu.edu.cn
  • 基金资助:
    国家自然科学基金(81972947)

Quantitative proteomic analysis of epidermal stem cells in oxygen-glucose deprivation conditions

ZHANG Huayu, YIN Siyuan, LIU Jian, MA Jiaxu, SONG Ru, CAO Guoqi, WANG Yibing   

  1. Department of Plastic Surgery, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan Clinical Research Center for Tissue Engineering Skin Regeneration and Wound Repair, Jinan 250014, Shandong, China
  • Published:2021-04-30

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摘要： 目的 寻找缺血条件下导致表皮干细胞功能紊乱的关键蛋白和通路,探讨慢性难愈合创面的形成机制。 方法 从新生乳鼠皮肤中提取表皮干细胞,在氧糖剥夺条件下分别培养0、3、6、9和12 h。将0 h作为对照组,其他各组为实验组,共得到4个比较组:OGD3_vs_OGD0、OGD6_vs_OGD0、OGD9_vs_OGD0和OGD12_vs_OGD0。使用串联质谱标记(TMT)技术对不同组别的表皮干细胞进行定量蛋白质组学测定,筛选4个比较组中发生明显差异表达的蛋白质,然后对这些差异表达蛋白进行基因本体(GO)类别、京都基因与基因组百科全书(KEGG)通路、蛋白质互作、基因重叠和专门针对线粒体自噬相关通路和蛋白的分析,以寻找在表皮干细胞功能紊乱中起关键作用的蛋白质和通路。 结果 共检测到4 852种可定量蛋白质,根据表达倍数>1.2倍和FDR值<0.05的筛选标准,4个比较组中分别筛选到上调的差异表达蛋白1种、225种、346种和386种,下调的差异表达蛋白26种、229种、330种和462种。通过对OGD6_vs_OGD0比较组的分析研究筛选出的关键类别和通路包括核糖核蛋白复合物的生物发生(GO:0022613),胞质核糖体大亚基(GO:0022625),染色质结合(GO:0003682)和核糖体(ko03010)。通过蛋白质互作分析筛选到21种hub蛋白,均为核糖体大亚基结构蛋白,而其中的seed蛋白为线粒体核糖体蛋白MRPL24;通过4个比较组的综合富集分析发现,各比较组的差异表达蛋白之间存在着大量的重叠,富集分析得到的关键通路都与蛋白质的生产加工有关;线粒体自噬相关的蛋白中,与溶酶体成熟相关的Tbc1d15、Rab7A和抑癌基因p53的表达量持续上调,与自噬流高低相关的LC3的表达量没有确切的变化趋势。 结论 通过TMT定量蛋白质组学分析获得的关键蛋白MRPL24和与蛋白质生产加工相关的富集通路是氧糖剥夺条件下表皮干细胞功能紊乱的关键,是进一步研究慢性难愈合创面形成机制的突破点,同时也为寻找相关治疗靶点提供了有力支持。

关键词: 慢性难愈合创面, 表皮干细胞, 氧糖剥夺, 线粒体自噬, 蛋白质组学

Abstract: Objective To find the key proteins and pathways leading to the dysfunction of epidermal stem cells in oxygen-glucose deprivation(OGD)conditions and to explore the formation mechanism of chronic hard healing wounds. Methods Epidermal stem cells were extracted from newborn mice and cultured normally to the P3 generation. The cells were cultured under OGD conditions for 0, 3, 6, 9 and 12 h, respectively, and then were divided into five goups: 0 h was taken as the control group, and the other groups were taken as the experimental groups. There are four comparison groups: OGD3_vs_OGD0, OGD6_vs_OGD0, OGD9_vs_OGD0 and OGD12_vs_OGD0. Quantitative proteomics determination was performed on different groups of epidermal stem cells using Tandem mass tag(TMT)technology. The four comparison groups were screened for proteins with significantly differential expression. These differentially expressed proteins(DEPs)were analyzed for Gene Ontology(GO)category, KEGG pathway, protein-protein interaction(PPI), gene overlap, and analysis of pathways and proteins related to mitophagy to find the key proteins and pathways in the dysfunction of epidermal stem cells. Results A total of 4 852 quantifiable proteins were detected in the experiment. In the four comparison groups, that is, OGD3_vs_OGD0, OGD6_vs_OGD0, OGD9_vs_OGD0, and OGD12_vs_OGD0, the number of up-regulated DEPs were 1, 225, 346 and 386, respectively, while the number of down-regulated DEPs were 26, 229, 330 and 462, respectively. In the analysis of the comparison group OGD6_vs_OGD0, key pathways included “ribonucleoprotein complex biogenesis(GO: 0022613)”“chromatin binding(GO: 0003682)”“cytoplasmic ribosomal large subunit(GO:0022625)” and “ribosome(ko03010)”,and the 21 hub proteins screened through PPI analysis were all structural proteins of the ribosome, of which the most important seed protein was the mitochondrial ribosomal protein MRPL24. In the comprehensive analysis of the four comparison groups, it was found that there was a large amount of overlap between the DEPs of each comparison group, and key pathways were all related to protein processing. In the analysis of mitophagy-related pathway and proteins, it was found that the expression of Tbc1d15, Rab7a, which were related to lysosomal maturation and p53, continued to be up-regulated, while the expression of LC3 related to the level of autophagy flow did not show a definite trend. Conclusion The key protein “MRPL24” obtained through TMT quantitative proteomics analysis and the pathways related to protein processing are the key to the dysfunction of epidermal stem cells in OGD conditions. They will be the breakthrough points for further research on the formation mechanism of chronic hard healing wounds.

Key words: Chronic hard healing wounds, Epidermal stem cells, Oxygen-glucose deprivation, Mitophagy, Proteomic analysis

中图分类号: 

  • R574
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