Effect and mechanism of mesenchymal stem cell derived exosomes in repair of tendon cell injury / 中华创伤杂志

Objective:To investigate the effects and mechanism of exosomes secreted by human umbilical cord-derived mesenchymal stem cells （hUC-MSCs） in repair of tendon cell injury.Methods:The hUC-MSCs which were stably subcultured were isolated and purified by a tissue block adherent method，and the immunophenotype of hUC-MSCs was detected by flow cytometry. The induction media was employed to induce the differentiation of hUC-MSCs to osteoblasts，chondroblasts and adipocytes，and cell identification was performed subsequently. The secreted exosomes of MSCs （MSCs-exosomes） were extracted using an ultracentrifugation method. The exosomes were detected by Western blot and electron microscopy，and the fusion ability of the exosome membrane was detected by PKH67 staining fluorescence. Forty Wistar rats were divided into tendon injury group （ n = 20） and normal group （ n = 20） according to the random number table. In tendon injury group，the rats were sacrificed with 100 mg/kg pentobarbital sodium one week after Achilles tendon transection，and the injured tendon cells were obtained following digestion of the Achilles tendon. In normal group，the rats were sacrificed without any treatment and the normal tendon cells were obtained concurrently. After the exosomes were co-cultured with tendon cells in vitro for 12，24，48，72 hours，the proliferation of tendon cells was detected by CCK-8 assay. After the tendon cells were treated with hUC-MSCs exosomes for 24 hours，the effects of exosomes on transforming growth factor β （TGF-β），bone morphogenetic protein （BMP），vascular endothelial growth factor （VEGF），fibroblast growth factor （FGF），interleukin（IL）-1β and tumor necrosis factor-α （TNF-α） were detected by Western blot，qPCR and immunofluorescence. Results:The hUC-MSCs were identified and hUC-MSCs-exosomes were isolated successfully. The cultured MSCs were fusiform and positive for Alanine aminopeptidase （CD13），integrin β-1 （CD29），ECTO-5'-nucleotidase （CD73），thymocyte surface antigen （CD90） and endothelin （CD105），but negative for human leukocyte DR antigen （HLA-DR），hematopoietic progenitor cell antigen （CD34） and leukocyte common antigen （CD45）. The exosomes isolated showed a round disc shape and a diameter of 30-100 nm with a depressed internal structure under the electron microscope which was verified via PKH67 staining and the motility-related protein-1 （CD9） and lysosomal associated membrane protein 3 （CD63） were highly expressed. The CCK-8 assay showed the cell viability in tendon injury group was markedly higher than that in normal group at 12 h，24 h，48 h，and 72 h following treatment of tendon cells （ P < 0.01）. The results of qPCR revealed that the mRNA expressions of TGF-β （1.850 ± 0.127），BMP （2.133 ± 0.398），FGF （1.610 ± 0.223） and VEGF （2.207 ± 0.059） in tendon injury group were markedly higher than those in normal group（1.004 ± 0.105，1.007 ± 0.145，1.007 ± 0.140，1.001 ± 0.065，respectively） （ P < 0.05）. However，the mRNA expressions of IL-1β （0.102 ± 0.009） and TNF-α （0.130 ± 0.013） in tendon injury group was markedly lower than those in normal group （1.004 ± 0.113，1.006 ± 0.134） （ P < 0.01）. The results of Western blot were consistent with those of qPCR. Conclusions:The exosomes secreted by hUC-MSCs can promote the growth of tendon cells and repair of tendon cell injury by up-regulating the expression of growth factors TGF-β，BMP，VEGF and FGF，and inhibiting the expression of inflammatory factors IL-1β and TNF-α.

Effect of decorin on proliferation and cell cycle of rabbit tendon cells in vitro / 第三军医大学学报

Objective To investigate effect of decorin on proliferation and cell cycle of rabbit tendon cells in vitro so as to explore the role of decorin in tendon wound healing.Methods Tendon cells derived from the tissue of rabbits flexor tendon were harvested and cultured in vitro with decorin of 0.25,1.25,2.5,5 ?g/ml.After culture of 12,24 or 48 h,the cell proliferation rate was measured by MTT colorimetric determination.After 24-hour culture with 0.25?g/ml decorin,the morphology of tendon cells was obtained and the cell cycle distribution was detected by flow cytometer.Results The proliferation of tendon cells was inhibited after 12-hour culture and significantly increased after 24-hour culture with 0.25,1.25,2.5 ?g/ml decorin.However,5 ?g/ml decorin could increase the proliferation after 12-hour culture,increase after 24-hour culture,with no significant difference between 24 h and 48 h.decorin at 0.25 ?g/ml could significantly increase the cells at S phase and PI after 24-hour culture(P