| Cutaneous injury caused by trauma,burns,chronic wounds,and skin wound infection,is one of the most common clinical manifestations,which gives rise to pain,psychological stress and loss of quality life.Numerous strategies,including growth factor,gene therapy,cell therapy,and skin grafts,have been applied to promote cutaneous wound healing in present clinical studies,whereas these therapies still suffer from potential concerns related to high cost,difficult preservation of drugs or donors,immune rejection,and secondary damage to the donor sites.Cutaneous wound healing generally includes three phases: hemostasis and inflammation,tissue formation,and tissue remodeling,which overlaps each other to a certain degree without a precisely defined period of time.During the above phases,cell migration is a common phenomenon and plays a key role in cell recruitment and promoting wound healing.In the migration process,the epithelial cells transform to mesenchymal-like cells,which is known as an epithelial-mesenchymal transition(EMT)process and allows rearranging the cytoskeleton,losing the cell-cell junctions and apical-basal polarity,enhancing motility and invasiveness,and finally promoting wound healing.Thus,it is significant to explore an effective way to induce EMT for increasing the migration of epithelial cells and further promoting cutaneous wound healing.In the second chapter,Vc-PEI CDots were prepared from ascorbic acid(Vc)and PEI by a one-step microwave-assisted heating method.After purification,chemical and biological properties of Vc-PEI CDots were detected.In the chemical properties,the high-resolution transmission electron microscope(HRTEM)images were taken.It was found that Vc-PEI CDots possess a uniform shape without apparent aggregation.The average diameter of CDots is 3.5 nm,and the lattice spacing is 0.21 nm.Vc-PEI CDots solution exhibits a strong photoluminescent(PL)emission,with the PL emission peak of our CDots centered at 533 nm.FTIR analysis and the XPS characterizations confirm the dehydration condensation and carbonization processes and the existence of carboxyl and amine groups in Vc-PEI CDots.For the biological properties,the results of MTT and apoptosis assay show that there is no significant difference in the proliferation and apoptosis of Ha Ca T cells treated by Vc-PEI CDots with concentrations ranging from 0 to 600 ?g/m L.After co-culturing with Vc-PEI CDots,Ha Ca T cells we observed under a confocal laser microscope and found that Vc-PEI CDots can be taken up by Ha Ca T cells and are mainly located around the cell surface and surrounding the cell.It was also proved that Vc-PEI CDots have the function of biological imaging.In this part,we synthesized Vc-PEI CDots and characterized its biological and chemical properties.It was proved that Vc-PEI CDot has good nano-sized structure,optical properties and biocompatibility.The surfaces of Vc-PEI CDots have abundant amino and carboxyl groups,which is beneficial for in vivo and in vitro experiments.In the third Chapter,we investigated the effect of Vc-PEI CDots inducing EMT on Ha Ca T cells.Ha Ca T cells were induced with different concentrations of Vc-PEI CDots,and with the concentration of Vc-PEI CDots increased,we observed a change in cell morphology from an epithelial phenotype to a mesenchymal phenotype.By Western blot,we demonstrated that the expression of mesenchymal molecular markers Fibronectin and Vimentin increased after Vc-PEI CDots inducing Haca T cells in a concentration-dependent way,while the expression of epithelial molecular marker E-cadherin decreased.We further verified the changes in the expression of EMT molecular markers by immunofluorescence.The result also showed that the expression of mesenchymal molecular marker Vimentin increased after Vc-PEI CDots inducing Ha Ca T cells,and the expression of molecular marker of epithelium E-cadherin decreased.We used scratch assay and Transwell assay to detect changes in the cell motility of Ha Ca T cells induced by Vc-PEI CDots and found that the cell motility of Ha Ca T cells was significantly enhanced with the concentration of Vc-PEI CDots increasing.Therefore,in this part,we demonstrated that Vc-PEI CDots can induce EMT on Ha Ca T cells.In the fourth chapter,we investigated the mechanism of Vc-PEI CDots-induced EMT in Ha Ca T cells.In order to clarify the relationship between Vc-PEI CDots and the TGF-? signaling pathway,we first performed experiments using the TGF-? receptor inhibitor and found that when TGF-? receptor was inhibited,Vc-PEI CDots could no longer promote the cell motility of Ha Ca T cells.To further detect the role of downstream signaling pathways,Western blot was used to test the effects of Vc-PEI CDots on canonical and non-canonical pathways in the TGF-? signaling pathway at different time points.As a result,it was found that Vc-PEI CDots can activate p38 protein in non-canonical pathway downstream of the TGF-? signaling pathway.We also found that the expression of the EMT transcription factor Snail increased with the inducing of Vc-PEI CDots.To further reveal the role of p38 in Vc-PEI CDots-induced EMT and the relationship between p38 and Snail,we conducted experiments using p38 inhibitors.We found that when p38 was inhibited,Vc-PEI CDots could no longer induce Ha Ca T cells to undergo EMT at the molecular level,and could not promote the cell motility of Ha Ca T cells and Snail expression.In this part,we demonstrated that Vc-PEI CDots induces EMT in Ha Ca T cells by activating the TGF-?/p38/Snail signaling pathway.In the fifth chapter,based on the ability of Vc-PEI CDots inducing EMT in epithelial cells that could promote cell motility,we investigated the role of Vc-PEI CDots in promoting skin wound healing in vivo.We selected male mice,prepared full-thickness skin wounds on the back after hair removal,and injected Vc-PEI CDots every 3 days.After 21 days of observation,we compared the Vc-PEI CDots group with the control group.There was a noticeable increase in healing speed on the 7th day in Vc-PEI CDots group,and the final results also showed that Vc-PEI CDots could significantly accelerate wound healing.The wound tissue was taken on day 7,15,and 21,stained with HE sections,and observed.The results showed that the Vc-PEI CDots group had better wound healing and less scar formation than the control group.On day 21,hearts,livers,spleens,and kidneys of the mice were removed for HE staining,and there was no significant difference in the cell morphology of the Vc-PEI CDots group compared with the control group.This part of the experiment shows that Vc-PEI CDots can accelerate skin wound healing in vivo,improve the quality of skin healing,and have good biological safety.In summary,we showed that Vc-PEI CDots synthesized by ascorbic acid and polyethyleneimine by one-step microwave-assisted heating method have rich carboxyl and amino groups and good biocompatibility.Vc-PEI CDots can induce EMT in Ha Ca T cells by activating the TGF-?/p38/Snail signaling pathway,thereby enhancing cell motility.In vivo experiments showed that Vc-PEI CDots can accelerate the migration of epithelial cells in full-thickness skin wounds by inducing EMT,so that the epidermal barrier could cover granulation tissue in wound area,prevents external stimulation,reduces inflammation and granulation tissue proliferation,and reduces scar formation,and finally promoting wound healing. |
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