中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (9): 616-623.doi: 10.3760/cma.j.issn.0412-4030.2019.09.006

• 论著 • 上一篇    下一篇

人脂肪干细胞源外泌体对HaCaT细胞损伤修复及迁移功能影响的初步实验研究

张远远1    曾悦2    朱艳霞2    廉翠红1   

  1. 1深圳大学第一附属医院  深圳市第二人民医院皮肤科  518035；2深圳大学医学部医学细胞生物学与遗传学系  518000
  • 收稿日期:2018-12-27 修回日期:2019-07-08 出版日期:2019-09-15 发布日期:2019-08-30
  • 通讯作者: 廉翠红；朱艳霞 E-mail:15814692161@163.com; yanxiazhu@szu.edu.cn
  • 基金资助:
    广东省医学科研基金（A2018555）；深圳市科技计划资助项目（JCYJ20170818100526471）

Effect of exosomes from human adipose-derived stem cells on wound healing and migration potential of HaCaT cells: a preliminary experimental study

Zhang Yuanyuan1, Zeng Yue2, Zhu Yanxia2, Lian Cuihong1   

  1. 1Department of Dermatology, The First Affiliated Hospital of Shenzhen University, The Second People′s Hospital of Shenzhen, Shenzhen 518035, China; 2Department of Medical Cell Biology and Genetics, Shenzhen University Health Science Center, Shenzhen 518000, China
  • Received:2018-12-27 Revised:2019-07-08 Online:2019-09-15 Published:2019-08-30
  • Contact: Lian Cuihong; Zhu Yanxia E-mail:15814692161@163.com; yanxiazhu@szu.edu.cn
  • Supported by:
    Medical Scientific Research Foundation of Guangdong Province of China（A2018555）; Shenzhen Science and Technology Plan Project （JCYJ20170818100526471）

PDF

5

赞

摘要： 【摘要】 目的 初步探究人脂肪干细胞源外泌体对表皮细胞迁移功能的影响。方法 从正常人抽脂手术中获取的脂肪组织分离培养脂肪干细胞，采用超速离心和超滤结合的改良法分离收集细胞上清液中的外泌体，采用透射电镜、动态光散射法以及Western印迹法鉴定分离出的外泌体。采用0、50、100、200、250、300、400、500、600、800 μmol/L H2O2分别作用HaCaT细胞1 h，CCK8法检测H2O2对HaCaT细胞存活率的影响。将HaCaT细胞分为正常组（无预处理）和损伤组（用100 μmol/L H2O2损伤预处理0.5 h），再将两组分别分为对照组和治疗组，治疗组与外泌体共培养，对照组不与外泌体共培养。用共聚焦荧光显微镜观察PKH26荧光标记的外泌体是否能被细胞吞噬，用划痕实验观察HaCaT细胞修复率，用Transwell实验评估细胞迁移能力。多组间比较采用单因素方差分析，两组间比较采用LSD检验，相关性检验采用Spearman检验。结果 透射电镜观察显示，分离所得的脂肪干细胞源外泌体是直径60 ~ 80 nm的茶托样纳米囊泡，而动态光散射法测量显示，分离的外泌体纯度为65.88%，表达CD63、Alix和TSG101，具有外泌体的基本特征。CCK8法显示，0 ~ 800 μmol/L H2O2处理HaCaT细胞1 h后细胞存活率呈逐渐降低趋势，H2O2浓度与HaCaT细胞存活率呈负相关（r = -0.91，P < 0.01）。共聚焦荧光显微镜观察，分离的外泌体能被损伤后的HaCaT细胞摄入。划痕实验显示，正常对照组、正常治疗组、损伤对照组、损伤治疗组HaCaT细胞12 h划痕面积修复率分别是40.26% ± 0.64%、69.57% ± 0.69%、32.28% ± 0.31%、69.62% ± 1.68%，损伤治疗组显著高于损伤对照组（t = 37.33，P < 0.01）。Transwell实验结果显示，4组10倍视野下迁移细胞数分别是20.85 ± 4.84、44.8 ± 5.24、14.95 ± 2.58、40.05 ± 7.66，损伤治疗组大于损伤对照组（t = 25.10，P < 0.01）。结论 人脂肪干细胞源外泌体具有促进氧化应激损伤的HaCaT细胞迁移的能力。

关键词: 干细胞, 皮下脂肪, 外泌体, 角蛋白细胞, 伤口愈合, 细胞迁移分析

Abstract: 【Abstract】 Objective To preliminarily evaluate the effect of exosomes from human adipose-derived stem cells （hADSCs） on the migration of epidermal cells. Methods hADSCs were isolated from adipose tissues obtained from a healthy woman after liposuction, and subjected to culture. Exosomes were collected from the culture supernatant of hADSCs by using a modified method combining ultracentrifugation with ultrafiltration, and identified and isolated by transmission electron microscopy （TEM）, dynamic light scattering （DLS） and Western blot analysis. Some cultured HaCaT cells were divided into several groups to be treated with hydrogen peroxide （H2O2） at different concentrations of 0, 50, 100, 200, 250, 300, 400, 500, 600, 800 μmol/L for 1 hour, and cell counting kit-8 （CCK8） assay was performed to evaluate the effect of H2O2 on the survival rate of HaCaT cells. Some HaCaT cells were classified into 2 groups to be pretreated with phosphate-buffered saline （PBS） （normal group） or 100 μmol/L H2O2 （injury group） for 0.5 hour; then, HaCaT cells in the 2 groups were separately divided into treatment group and control group to be co-cultured with exosomes or not. Confocal fluorescence microscopy was conducted to confirm the uptake of PKH26-labelled exosomes by HaCaT cells, scratch assay to estimate the wound healing potential, and Transwell assay to evaluate the migratory activity of HaCaT cells. Statistical analysis was carried out by using one-way analysis of variance for comparison among groups, least significant difference （LSD）-t test for multiple comparisons, and Spearman correlation analysis for analyzing correlations. Results TEM showed that the exosomes isolated from hADSCs were saucer-like nanovesicles with diameters of 60 - 80 nm. DLS revealed that the purity of the isolated exosomes was 65.88%, and they were stained positively for CD63, Alix and TSG101, which coincided with the basic characteristics of exosomes. CCK8 assay showed that survival rates of HaCaT cells gradually decreased along with the increase of H2O2 concentrations after 1-hour treatment, and were negatively correlated with the concentration of H2O2 （r = -0.91, P < 0.01）. Confocal fluorescence microscopy showed that the isolated exosomes could be endocytosed into impaired HaCaT cells. Scratch assay showed that the gap-filling rates at 12 hours were 40.26% ± 0.64%, 69.57% ± 0.69%, 32.28% ± 0.31% and 69.62% ± 1.68% in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and the injury treatment group showed a significantly increased gap-filling rate compared with the injury control group （t = 37.33, P < 0.01）. Transwell assay showed that the number of migratory cells per × 10 field was 20.85 ± 4.84, 44.8 ± 5.24, 14.95 ± 2.58 and 40.05 ± 7.66 in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and was significantly larger in the injury treatment group than in the injury control group （t = 25.10, P < 0.01）. Conclusion Exosomes isolated from hADSCs can improve the migration of HaCaT cells after oxidative injury.

Key words: Stem cells, Subcutaneous fat, Exosomes, Keratinocytes, Wound healing, Cell migration assays