成纤维细胞与间充质干细胞来源外泌体修复急性皮肤创面的比较

李锐阳; 董秋霖; 胡金华; 姜美花

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0302

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成纤维细胞与间充质干细胞来源外泌体修复急性皮肤创面的比较

基础研究 | 更新时间：2024-02-19

- 成纤维细胞与间充质干细胞来源外泌体修复急性皮肤创面的比较
- Comparison of Exosomes Derived from Fibroblasts and Mesenchymal Stem Cells for the Treatment of Acute Wound Healing
- 中山大学学报（医学科学版） 2023年44卷第3期页码：369-378
- 作者机构：
  
  1.中山大学中山医学院干细胞与组织工程研究中心，广东广州 510080
  2.广州医科大学附属广州市妇女儿童医疗中心泌尿外科，广东广州 510120
- 作者简介：
  
  李锐阳，硕士生，研究方向：干细胞与再生医学，E-mail：449692853@qq.com
- 基金信息：
  
  国家自然科学基金(31972894)
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0302
  中图分类号： R64
- 纸质出版日期：2023-05-20，
  
  收稿日期：2023-02-10，
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李锐阳,董秋霖,胡金华等.成纤维细胞与间充质干细胞来源外泌体修复急性皮肤创面的比较[J].中山大学学报（医学科学版）,2023,44(03):369-378.

LI Rui-yang,DONG Qiu-lin,HU Jin-hua,et al.Comparison of Exosomes Derived from Fibroblasts and Mesenchymal Stem Cells for the Treatment of Acute Wound Healing[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(03):369-378.
李锐阳,董秋霖,胡金华等.成纤维细胞与间充质干细胞来源外泌体修复急性皮肤创面的比较[J].中山大学学报（医学科学版）,2023,44(03):369-378. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0302.

LI Rui-yang,DONG Qiu-lin,HU Jin-hua,et al.Comparison of Exosomes Derived from Fibroblasts and Mesenchymal Stem Cells for the Treatment of Acute Wound Healing[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(03):369-378. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0302.

摘要

目的

本研究旨在比较成纤维细胞来源外泌体与间充质干细胞来源外泌体对小鼠急性皮肤创面的修复作用。

方法

体外分离培养原代人真皮成纤维细胞（hDF）并鉴定。用超速离心法提取人真皮成纤维细胞来源外泌体（hDF-EXO）、人骨髓间充质干细胞来源外泌体（hMSC-EXO）并鉴定；两种外泌体与hDF共培养24 h后，CCK8细胞增殖实验和划痕实验检测细胞增殖和迁移活性；采用8周龄雌性C57BL/6小鼠构建急性全层皮肤切除模型，在伤口上分别局部涂敷PBS（对照组）、hDF-EXO、hMSC-EXO进行治疗。术后第0、2、4、7天观察小鼠伤口并计算伤口面积。术后第1天取伤口组织检测对外泌体摄取情况，并利用qPCR检测伤口组织TNF-α、IL-6、IL-1β、IL-10炎症因子水平。术后第7天取伤口组织进行HE染色观察伤口组织结构变化，免疫荧光染色观察伤口组织PDGFR-α、α-SMA、Ki67表达情况。

结果

hDF表面分子及特异性标志物表达符合成纤维细胞特性。hDF-EXO和hMSC-EXO的形状、粒径、标志物均符合外泌体鉴定标准，且两者浓度差异无统计学意义（

0.05）；体外试验数据显示，CCK8法检测显示hDF-EXO组和hMSC-EXO组的细胞活力均显著高于对照组（

0.01），且hDF-EXO组的细胞活力明显高于hMSC-EXO组（

0.01）。划痕实验显示， hDF-EXO组24 h划痕面积修复率显著高于对照组（

0.01）和hMSC-EXO组（

0.05）；体内试验数据显示，术后第1天，观察到两种外泌体均被移植周围组织摄入。qPCR检测hDF-EXO组和hMSC-EXO组伤口组织TNF-α、IL-6、IL-1β炎症因子水平均显著低于对照组（均

0.01），且hMSC-EXO组炎症因子水平低于hDF-EXO组（均

0.01）。术后第7天hDF-EXO组和hMSC-EXO组伤口面积均显著小于对照组（均

0.01），且hDF-EXO组伤口面积小于hMSC-EXO组（

0.05）。HE染色显示，与对照组相比，hMSC-EXO组小鼠伤口残留血痂，表皮不连续，仅有部分新生纤维组织。hDF-EXO组小鼠实现了更快的伤口再上皮化，表皮连续性好，新生纤维组织排列整齐。免疫荧光染色显示，hDF-EXO组和hMSC-EXO组相比对照组，成纤维细胞、肌成纤维细胞及增殖细胞数量显著增多，且hDF-EXO组明显多于hMSC-EXO组。

结论

两种外泌体均能有效促进创面愈合，hDF-EXO通过显著促进成纤维细胞增殖、迁移以及分化为肌成纤维细胞（FMT）加速伤口修复，且其修复作用优于hMSC-EXO。而hMSC-EXO相较于hDF-EXO在伤口早期可发挥更佳的炎症抑制作用。

Abstract

Objective

To compare the therapeutical effect of exosomes derived from fibroblasts and mesenchymal stem cells on acute wound healing.

Methods

Primary human dermal fibroblasts （hDF） were isolated， cultured and identified. Human bone marrow mesenchymal stem cell exosomes （hMSC-EXO） and hDF exosomes （hDF-EXO） were extracted by ultracentrifuga tion. After 24 h of coincubation with hDF-EXO or hMSC-EXO， hDFs proliferation and migratory capacity were evaluated by cell counting kit-8 （CCK8） assay and scratch test. Full-thickness cutaneous wounds were created on 8-week-old female C57BL/6 mice， and topically applied with PBS （control）， hDF-EXO or hMSC-EXO. Wounds were measured at day 0， 2， 4， 7， and the uptake of exosomes in wound was observed at day 1. Quantitative PCR （qPCR） analysis was performed to detect the mRNA expression levels of TNF-α， IL-6， IL-1β， IL-10 in wound at day 1. HE staining was conducted to analyze the histological structure of wounds at day 7， while immunofluorescence staining was used to examine expression of PDGFR-α、α-SMA、Ki67.

Results

hDF exhibited certain fibrolast-like characteristics with respect to expression of cell surface markers and specific proteins. hDF-EXO and hMSC-EXO presented exosomal morphology， size， and markers， and both concentrations were not statistically different （

0.05）； CCK8 assay showed that both exosomes promoted hDF cell viability， compared with the negative control （

0.01）， and hDF-EXO group had greater cell viability than hMSC-EXO group （

0.01）. Scratch test indicated that hDF-EXO induced a significant increase in scratch healing rate versus the negative control

（P

0.01）， hMSC-EXO （

0.05）. In vivo experiments showed wound tissues took up exosomes at day 1. qPCR detected TNF-α， IL-6， IL-1β expression levels in wound at day 1 were lower in exosomes group than in the control group， and were the lowest in hMSC-EXO group （all

0.01）. Wound areas were measured smaller at day 7 in exosomes group than in the control group （all

0.01） and hDF-EXO group had better closure than hMSC-EXO group （

0.05）. HE staining revealed that compared with control group， scar， incomplete epidermis and few collagen deposition remained in the hMSC-EXO group， whereas hDF-EXO group showed re-epithelialization， continuous neo-epidermis and regenerated dermis. Immunofluorescence staining suggested that the number of fibroblasts， myofibroblasts， proliferating cells was higher in both exosomes group than that in the control group， especially the highest in hDF-EXO group.

Conclusion

Our study shows both exosomes accelerate wound healing， whereas hDF-EXO is more effective in promoting fibroblasts proliferation， migration， transition to myofibroblasts， and hMSC-EXO may play a role in inhibiting inflammatory reaction during early stage of wound healing.

关键词

外泌体伤口愈合成纤维细胞间充质干细胞

Keywords

exosomewound healingfibroblastmesenchymal stem cell

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