Effects And Mechanisms Of IPSCs-derived Microvesicles/Hsp90α On The Wound Healing Of Deep Second Degree Burn In Mice

Part one Effects and mechanisms of miPSCs-derived microvesicles on wound healing of deep second degree burns in mice(A)PurposeTo explore the effect and related mechanism of miPSC-derived micro vesicles(miPSCs-MVs)in the treatment of deep second degree burns in mice.(B)MethodsⅠ.Effects of miPSCs-MVs on wound healing of deep second degree burns in mice 1.Collect miPS cell conditioned medium to extract and identify extracellular vesicles.2.miPSCs-MVs were locally applied to the treatment of deep second degree burns in mouse skin.Observe wound healing and epithelialization by gross and HE staining.3.PKH-67 labeled miPSCs-MVs were applied to the treatment of deep second degree burn in mice skin.In vivo imaging and frozen sections were performed to observe the stability of miPSCs-MVs.Ⅱ.The signal mechanism of miPSCs-MVs to promote the wound healing of deep second degree burns in mice1.PKH-67 labeled miPSCs-MVs acted on HaCaT cells,and the uptake was observed under a microscope.The effects of miPSCs-MVs on cell proliferation and migration were observed by CCK-8,cell EdU experiments,and cell scratches.2.Screening miRNAs with high abundance and their effects on the migration of HaCaT cells through high-throughput miRNA sequencing,cell scratch experiments,and cell EdU experiments.3.Western blot,RT-qPCR,double luciferase reporter gene experiments,and cell scratch experiments confirmed that miR-16-5p targeted inhibition of Dsg3 regulated HaCaT cells migration via the p38 signaling pathway.Ⅲ.Effect of miR-16-5p on deep second degree burns in mouse skin1.miR-16-5p was applied to the treatment of deep second degree burns in mice.Observed wound healing and epithelialization by gross,morphological,tissue EdU staining and immunohistochemical staining.(C)Results1.The miPSCs-MVs have a double-film-forming structure with an average particle size of 214.6 nm.2.Compared with the PBS group,the wound healing effect and epithelialization of the miPSCs-MVs group were significantly enhanced.3.In vivo imaging of mice shows that the red signal area is obvious on the first day after burn in the miPSCs-MVs group.Frozen section microscopy showed a large amount of green fluorescence in the cytoplasmic area of the epithelial cells on the first day after burn in the miPSCs-MVs group.4.miPSCs-MVs can be ingested by HaCaT cells and promote HaCaT cells migration.5.miR-16-5p has the highest expression abundance in miPSCs-MVs and can promote the migration of HaCaT cells without significant effect on proliferation.6.miR-16-5p targeted inhibition of Dsg3 regulated HaCaT cells migration via the p38 signaling pathway.7.miR-16-5p significantly increased the wound healing rate and promoted the epithelialization process.(D)ConclusionmiPSCs-MVs promote wound healing by promoting the process of re-epithelialization of deep second degree burn wounds in mice.miR-16-5p in miPSCs-MVs targets the inhibition of Dsg3 to activate the p38 signaling pathway,thereby promoting cell migration.miR-16-5p can promote the healing of deep second degree burns in mice.Part two Hsp90α promotes the migration of iPSCs-derived keratinocyte to accelerate deep second degree burn wound healing in mice(A)PurposeTo investigate the effect and mechanism of transplantation of miPSC-derived keratinocytes combined with Hsp90α on the healing of second degree burn in mice skin.(B)Methods1.miPS cells were induced to differentiate into keratinocytes,and immunofluorescence staining was used to detect the expression of keratinocyte marker protein.2.PBS,miPSCs-KCs,Hsp90α,miPSCs-KCs combined with Hsp90α were injected into the deep abdominal second degree burn wounds.Observe wound healing and epithelialization by gross and HE staining.3.The CSFE-labeled miPSCs-KCs and the CSFE-labeled miPSCs-KCs plus Hsp90α were injected into the wound,respectively.Frozen sections were used to observe the miPSCs-KCs transplantation after the burn.4.The effect of Hsp90α on miPSCs-KCs migration was detected by Transwell experiment,and the change of Akt signaling pathway was detected by western blot.(C)Results1.miPSCs-KCs express keratinocyte marker protein.2.Compared with the PBS group,miPSCs-KCs group,and Hsp90α group,the wound area of miPSCs-KCs plus Hsp90α group was significantly reduced,and re-epithelialization was significantly enhanced.3.The CSFE-labeled miPSCs-KCs combined with Hsp90α group expressed more green fluorescence than the CSFE-labeled miPSCs-KCs group in the wound epithelialization site.4.Hsp90α can promote miPSCs-KCs migration and activate Akt signaling pathway(D)ConclusionHsp90α can promote the migration of miPSCs-KCs by activating the Akt signaling pathway,thereby enhancing the role of miPSCs-KCs transplantation in the treatment of deep second degree burn skin healing in mice.