Email Alerts
RSS
Volume 37 Issue 12
Dec.  2021
Turn off MathJax
Article Contents
Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2021  >  37(12): 1175-1184
Wei P,Xu ZR,Chen YM,et al.The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment[J].Chin J Burns,2021,37(12):1175-1184.DOI: 10.3760/cma.j.cn501120-20210424-00154.
Citation: Wei P,Xu ZR,Chen YM,et al.The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment[J].Chin J Burns,2021,37(12):1175-1184.DOI: 10.3760/cma.j.cn501120-20210424-00154.
shu

The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment

doi: 10.3760/cma.j.cn501120-20210424-00154

  • Department of Burns and Wound Repair, Union Hospital, Fujian Medical University, Fujian Provincial Burn Research Institute, Fujian Burn Medical Center, Fujian Provincial Key Laboratory of Burn and Trauma, Fuzhou 350001, China

Funds:

The Construction Program of Medical "Double High" in Fujian Province of China 202176

More Information
  • Corresponding author: Chen Zhaohong, Email: doctorczh@163.com
  • Received Date: 2021-04-24
    Abstract
  •   Objective  To investigate the effect and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment.  Methods  The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy pregnant women at full term delivery in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019, and the primary human amniotic epithelial cells (hAECs) were isolated. The growth status and morphological changes of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed, and the expressions of the cells surface markers of CD73, CD90, CD29, CD34, and human leukocyte antigen DR (HLA-DR). The 2nd to 4th passages of hAECs were used in the following experiments. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptake of hAEC-Exos by HaCaT was observed by inverted fluorescence microscopy. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group, and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation activity after 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test.  Results  Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and HLA-DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in PBS group were significantly lower than those in hAEC-Exos group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group (t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group (t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group (t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group (t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar (P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group (t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group (t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group (t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group (t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly more than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively (t=7.464, 1.232, P<0.01).  Conclusions  PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.

     

    • FullText(HTML)
  • loading
    • References(27)
  • [1]
    BurgessJL,WyantWA,Abdo AbujamraB,et al.Diabetic wound-healing science[J].Medicina (Kaunas),2021,57(10):1072. DOI: 10.3390/medicina57101072.
    [2]
    RatajczakJ,WysoczynskiM,HayekF,et al.Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication[J].Leukemia,2006,20(9):1487-1495.DOI: 10.1038/sj.leu.2404296.
    [3]
    FerreiraADF, GomesDA. Stem cell extracellular vesicles in skin repair[J].Bioengineering (Basel),2018,6(1):4. DOI: 10.3390/bioengineering6010004.
    [4]
    van NielG, D'AngeloG, RaposoG.Shedding light on the cell biology of extracellular vesicles[J].Nat Rev Mol Cell Biol,2018,19(4):213-228. DOI: 10.1038/nrm.2017.125.
    [5]
    ZhangW,BaiX,ZhaoB,et al.Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K/Akt signaling pathway[J].Exp Cell Res,2018,370(2):333-342. DOI: 10.1016/j.yexcr.2018.06.035.
    [6]
    ZhangJ,ChenC,HuB,et al.Exosomes derived from human endothelial progenitor cells accelerate cutaneous wound healing by promoting angiogenesis through Erk1/2 signaling[J].Int J Biol Sci,2016,12(12):1472-1487.DOI: 10.7150/ijbs.15514.
    [7]
    LiX, LiuL, YangJ, et al. Exosome derived from human umbilical cord mesenchymal stem cell mediates miR-181c attenuating burn-induced excessive inflammation[J]. EBioMedicine, 2016,8:72-82. DOI: 10.1016/j.ebiom.2016.04.030.
    [8]
    ZhaoB,LiX,ShiX,et al.Exosomal microRNAs derived from human amniotic epithelial cells accelerate wound healing by promoting the proliferation and migration of fibroblasts[J].Stem Cells Int,2018,2018:5420463.DOI: 10.1155/2018/5420463.
    [9]
    ZhengY,ZhengS,FanX,et al.Amniotic epithelial cells accelerate diabetic wound healing by modulating inflammation and promoting neovascularization[J].Stem Cells Int,2018,2018:1082076.DOI: 10.1155/2018/1082076.
    [10]
    ZhengY,JiS,WuH,et al.Topical administration of cryopreserved living micronized amnion accelerates wound healing in diabetic mice by modulating local microenvironment[J].Biomaterials,2017,113:56-67.DOI: 10.1016/j.biomaterials.2016.10.031.
    [11]
    WeiP,ZhongC,YangX,et al.Exosomes derived from human amniotic epithelial cells accelerate diabetic wound healing via PI3K-AKT-mTOR-mediated promotion in angiogenesis and fibroblast function[J/OL].Burns Trauma,2020,8:tkaa020[2021-04-24].https://pubmed.ncbi.nlm.nih.gov/32923490/.DOI: 10.1093/burnst/tkaa020.
    [12]
    XiaoGY,ChengCC,ChiangYS,et al.Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy[J].Sci Rep,2016,6:23120.DOI: 10.1038/srep23120.
    [13]
    FortunatoO,GaspariniP,BoeriM,et al.Exo-miRNAs as a new tool for liquid biopsy in lung cancer[J].Cancers (Basel),2019,11(6):888. DOI: 10.3390/cancers11060888.
    [14]
    HuY,RaoSS,WangZX,et al.Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function[J].Theranostics,2018,8(1):169-184.DOI: 10.7150/thno.21234.
    [15]
    HuangH,CuiW,QiuW,et al.Impaired wound healing results from the dysfunction of the Akt/mTOR pathway in diabetic rats[J].J Dermatol Sci,2015,79(3):241-251.DOI: 10.1016/j.jdermsci.2015.06.002.
    [16]
    CastilhoRM,SquarizeCH,GutkindJS.Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing[J].Oral Dis,2013,19(6):551-558.DOI: 10.1111/odi.12070.
    [17]
    ShawTJ,MartinP.Wound repair: a showcase for cell plasticity and migration[J].Curr Opin Cell Biol,2016,42:29-37.DOI: 10.1016/j.ceb.2016.04.001.
    [18]
    HuSC,LanCE.High-glucose environment disturbs the physiologic functions of keratinocytes: focusing on diabetic wound healing[J].J Dermatol Sci,2016,84(2):121-127.DOI: 10.1016/j.jdermsci.2016.07.008.
    [19]
    ZhaoB,LiuJQ,YangC,et al.Human amniotic epithelial cells attenuate TGF-β1-induced human dermal fibroblast transformation to myofibroblasts via TGF-β1/Smad3 pathway[J].Cytotherapy,2016,18(8):1012-1024.DOI: 10.1016/j.jcyt.2016.04.009.
    [20]
    ZhaoB,LiuJQ,ZhengZ,et al.Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways[J].Cell Tissue Res,2016,365(1):85-99.DOI: 10.1007/s00441-016-2366-1.
    [21]
    CamussiG,DeregibusMC,BrunoS,et al.Exosomes/microvesicles as a mechanism of cell-to-cell communication[J].Kidney Int,2010,78(9):838-848.DOI: 10.1038/ki.2010.278.
    [22]
    FriedmanRC,FarhKK,BurgeCB,et al.Most mammalian mRNAs are conserved targets of microRNAs[J].Genome Res,2009,19(1):92-105.DOI: 10.1101/gr.082701.108.
    [23]
    MengZ,ZhouD,GaoY,et al.miRNA delivery for skin wound healing[J].Adv Drug Deliv Rev,2018,129:308-318.DOI: 10.1016/j.addr.2017.12.011.
    [24]
    MulhollandEJ,DunneN,McCarthyHO.MicroRNA as therapeutic targets for chronic wound healing[J].Mol Ther Nucleic Acids,2017,8:46-55.DOI: 10.1016/j.omtn.2017.06.003.
    [25]
    FahsF, BiX, YuFS, et al. New insights into microRNAs in skin wound healing[J]. IUBMB Life, 2015,67(12):889-896. DOI: 10.1002/iub.1449.
    [26]
    LiD,LiXI,WangA,et al.MicroRNA-31 promotes skin wound healing by enhancing keratinocyte proliferation and migration[J].J Invest Dermatol,2015,135(6):1676-1685.DOI: 10.1038/jid.2015.48.
    [27]
    DeppeJ,SteinritzD,SantovitoD,et al.Upregulation of miR-203 and miR-210 affect growth and differentiation of keratinocytes after exposure to sulfur mustard in normoxia and hypoxia[J].Toxicol Lett,2016,244:81-87.DOI: 10.1016/j.toxlet.2015.09.012.
    • Relative Articles
    • Supplements(0)
    • Cited By
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(8)  / Tables(6)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (420) PDF downloads(46) Cited by()
    Proportional views
    Related

    Copyright © Chinese Journal of Burns京ICP备07035254号-14

    E-mail：shaoshangzazhi@163.com

    Website：https://zhsszz.xml-journal.net/

    Supported by: Beijing Renhe Information Technology Co. Ltd    

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return