关键词: 伤口愈合, 间质干细胞, 脐带, 外泌体, 成纤维细胞, 胶原Ⅰ型, 纤连蛋白类, 血管内皮生长因子类

Abstract: 【Abstract】 Objective To investigate the role of umbilical cord mesenchymal stem cell-derived exosomes （ucMSC-exos） in acute skin wound healing in mice. Methods ucMSC-exos were extracted by ultracentrifugation, and identified by transmission electron microscopy, Western blot analysis of exosome surface markers CD63 and TSG101, and particle size analysis. Firstly, in vitro cultured third- to fifth-passage human skin fibroblasts （HSF） were incubated with high-glucose Dulbecco′s modified Eagle′s medium （DMEM） containing 0, 1 and 2 μg/ml exosome suspension for 24 hours （negative control group, 1- and 2-μg/ml groups, respectively）, and cell counting kit-8 （CCK8） assay was performed to evaluate the effect of ucMSC-exos on the proliferative activity of HSF. Secondly, 24 male BALB/c mice aged 8 weeks were selected to construct a mouse model of full-thickness skin wound, and then divided into ucMSC-exos group and phosphate-buffered saline （PBS） group by using a random number table to be subcutaneously injected with exosome suspension and PBS respectively at multiple equidistant sites located about 1 mm apart from the wound edge. On days 0, 4, 7, 10 and 14 after operation, the wounds in mice were observed, and the percentage of residual wound area was calculated in the above two groups. On days 7 and 14 after operation, wound tissues were resected and subjected to hematoxylin and eosin （HE） and Masson staining to observe structural changes of skin tissues. On day 14 after operation, wound tissues were collected in the two groups, and real-time quantitative PCR （qRT-PCR） and Western blot analysis were performed to determine the mRNA and protein expression of type Ⅰ collagen, fibronectin and vascular endothelial growth factor, respectively. Statistical analysis was carried out by using one-way analysis of variance, least significant difference-t test, two-way repeated measures analysis of variance and unpaired t-test. Results Under the transmission electron microscope, the ucMSC-exos were oval in shape with a diameter of about 100 nm; Western blot analysis showed positive expression of ucMSC-exos surface proteins CD63 and TSG101; particle size analysis showed that 96.2 % of the ucMSC-exos had diameters of 30 - 150 nm. CCK8 assay showed that the relative proliferative activity of HSF was significantly higher in the 1- and 2-μg/ml groups （0.97 ± 0.05, 1.08 ± 0.07, respectively） than in the negative control group （0.71 ± 0.04; t = 2.00, 7.05, respectively, both P < 0.05）, and significantly higher in the 2-μg/ml group than in the 1-μg/ml group （t = 5.09, P < 0.05）. On days 4, 7, 10 and 14 after operation, the percentage of residual wound area was significantly lower in the ucMSC-exos group than in the PBS group （all P < 0.05）. HE and Masson staining showed increased numbers of hair follicles, glands and granulation tissues, more neovascularization, and neater arrangement of collagens in neonatal skin tissues of the mice in the ucMSC-exos group compared with the PBS group. qRT-PCR and Western blot analysis showed significantly increased mRNA and protein expression of type Ⅰ collagen, fibronectin and vascular endothelial growth factor in the ucMSC-exos group compared with the PBS group （all P < 0.01）. Conclusion Subcutaneous injections of ucMSC-exos can promote acute skin wound healing in mice, likely by promoting the synthesis of extracellular matrix and vascular endothelial growth factor in wound tissues of mice and proliferation of HSF.

Key words: Wound healing, Mesenchymal stem cells, Umbilical cord, Exosomes, Fibroblasts, Collagen type I, Fibronectins, Vascular endothelial growth factors