月经血源性间充质干细胞来源外泌体对SD大鼠皮肤创面愈合的促进作用及其机制

Promoting effect of menstrual blood-derived mesenchymal stem cells-derived exosomes on skin wound healing in SD rats and its mechanism

目的观察月经血源性间充质干细胞(MenSCs)中分离的外泌体(MenSC-Exos)对SD大鼠皮肤创面愈合的促进作用,并探讨其机制。方法分离培养健康成年女性志愿者的MenSCs,提取MenSC-Exos;将SD大鼠随机分为实验组和对照组,制作背部皮肤机械创面,分别给予MenSC-Exos或PBS治疗;观察创面愈合情况并计算创面愈合率,采用HE染色观察创面炎症细胞浸润情况,免疫荧光法检测M1、M2型巨噬细胞,采用免疫组化法检测创面组织中的白细胞介素(IL)-6、IL-10、新生血管、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白,采用Masson染色检测创面胶原蛋白含量及成熟度。培养成纤维细胞并分成外泌体组和PBS组,分别加入MenSC-Exos或PBS,采用CCK-8法检测细胞增殖能力,划痕实验检测细胞迁移能力。结果实验组术后第7、14天创面愈合率、Ⅲ型胶原蛋白沉积率和胶原蛋白含量高于对照组,Ⅰ型胶原蛋白沉积率低于对照组(P均<0.05)。实验组术后第7天创面炎症细胞数量和M1/M2低于对照组,M2型巨噬细胞数量高于对照组(P均<0.05);与对照组相比,实验组术后第3、7天创面IL-6表达降低,IL-10表达及血管内皮细胞表达升高(P均<0.05)。外泌体组培养1~5 d细胞增殖能力均高于PBS组,外泌体组培养24 h细胞迁移率高于PBS组(P均<0.05)。结论MenSC-Exos能够促进大鼠皮肤创面愈合,机制可能与调节炎症反应、促进血管生成和成纤维细胞增殖、调节胶原重塑有关。

Objective To observe the promotional effect of exosomes(MenSC-Exos)isolated from menstrual bloodderived mesenchymal stem cells(MenSCs)on the healing of skin wounds in SD rats and to investigate the mechanism.Methods MenSCs from healthy adult female volunteers were isolated and cultured,and MenSC-Exos were extracted.SD rats were randomly divided into the experimental and control groups,and mechanical wounds of the dorsal skin were made and treated with MenSC-Exos or PBS,respectively.We observed the wound healing and calculated the wound healing rate,used HE staining to observe the infiltration of inflammatory cells on the wound,used immunofluorescence to detect M1 and M2 macrophages,used immunohistochemistry to detect IL-6,IL-10,neovasculature,collagen type I,collagen type III in the tissues of the wound,and used Masson staining to detect the content and maturity of collagen on the wound.Fibroblasts were cultured and divided into the exosome and PBS groups,during which MenSC-Exos or PBS were added,respectively,and cell proliferation ability was detected by CCK-8 assay and cell migration ability by Scratch assay.Results The wound healing rate,type III collagen deposition rate,and collagen content of the experimental group were higher than those of the control group on postoperative day 7 and 14,and the type I collagen deposition rate was lower than that of the control group(all P<0.05).The number of inflammatory cells and the M1/M2 ratio of the trauma on postoperative day 7 were lower in the experimental group than in the control group,and the number of M2-type macrophages was higher than that in the con‐trol group(all P<0.05).Compared with the control group,the experimental group had lower IL-6 expression and higher IL-10 expression and vascular endothelial cell expression on the 3rd and 7th postoperative days of the trauma(all P<0.05).The cell proliferation ability of the exosome group was higher than that of the PBS group from 1 to 5 days of culture,and the cell migration rate of the exosome group was higher than that of the PBS group at 24 h of culture(all P<0.05).Conclusion MenSC-Exos can promote the healing of rat skin wounds,and the mechanism may be related to the regulation of inflammato‐ry response,promotion of angiogenesis and fibroblast proliferation,and regulation of collagen remodeling.