Proteomics Analysis On The Mechanism Of Repair Of Radiation-induced Skin Ulcers Treated With Human Umbilical Cord Mesenchymal Stem Cells

Radiation ulcer is a common skin radiation injury,mainly in malignant tumor radiotherapy,wartime nuclear radiation,accident exposure and so on.As a result of wound necrosis and loss of tissue fluid,malnutrition and infection,disability rate and mortality increased year by year.In recent years,high pressure oxygen therapy and VSD negative pressure blocking and suction have been widely used in the treatment of chronic refractory wounds,but the therapeutic effect on radiation skin ulcers is poor or even ineffective.Because the chronic radiation ulcer wound is characterized by prolonged,potential,progressive.Especially at present,the mechanism of radiation-induced skin ulcer formation and refractory healing is still unclear.Therefore,it is urgent to explore the mechanism of radiation ulcer healing and to find a more safe and effective treatment method.Human cord mesenchymal stem cells have the biological characteristics of homing,differentiation,paracrine and so on.It enables MSCs to repair chronic skin ulcer caused by radiation skin damage,diabetes,arterial occlusive vasculitis and bedsore.Human umbilical cord mesenchymal stem cells were isolated and cultured from the umbilical cord after delivery.The source is convenient and the immunogenicity is low,which may become the main seed cell for repairing chronic skin ulcer.Proteomics research based on iTRAQ technology is helpful to explore the pathogenesis of disease.At present,the mechanism of radiation-induced skin ulcer and the mechanism of mesenchymal stem cells repairing chronic skin ulcer have not been reported.Liquid chromatography-mass spectrometry(LC-MS/MS)combined with iTRAQ technology can display the changes of protein expression and the total appearance of protein.It has become an effective tool for screening differentially expressed proteins.In this study,the adherent growth of hUCMSCs was obtained by mechanical method from the umbilical cord warton glue,and cultured,subcultured,amplified and identified to provide seed cells for the next experiment.Secondly,the animal model of radiation skin ulcer was established,and hUCMSCs was applied to the wound of skin ulcer.The ability of homing and the therapeutic effect were observed.Thirdly,iTRAQ-LC-MS/MS technique was used to find the differential proteins between the tissue of radiation skin ulcer and the normal skin and the treatment group of hUCMSCs.4th.The differential proteins screened by proteomics were verified by Western blot or immunohistochemistry to investigate the mechanism of radiation ulcer formation and refractory healing.And to find out the repair mechanism of hUCMSCs transplantation to repair chronic skin wounds.The results of in vivo study showed that the adherent growth of hUCMSCs could be obtained from the umbilical cord warton glue by mechanical method,which could meet the needs of subsequent experiments through subculture,culture and proliferation.HUCMSCs can homing,proliferating and accelerating wound healing and improving the quality of skin repair in radiation skin ulcer tissue.Proteomics studies based on iTRAQ technique showed that the differential proteins were mainly keratin and proteins related to cell apoptosis and cell proliferation.Some differentially expressed proteins were further verified by Western blot or immunohistochemistry.Enrichment analysis of KEGG pathway showed that p53 cell signaling pathway,PI3K-Akt cell signal pathway,MAPK cell signaling pathway and Wnt signaling pathway are involved in the repair of radiation skin ulcer by human umbilical cord mesenchymal stem cells.This may be related to the paracrine effect of hUCMSCs in promoting the proliferation of basal stem cells in radiation-induced skin ulcer tissues and inhibiting cell apoptosis.These results provide new ideas and methods for mesenchymal stem cells to repair chronic skin wounds.This experiment is divided into four parts,the purpose,methods,results and conclusions are as follows.Part ?: Extraction,identification and biological characteristics of human umbilical cord mesenchymal stem cellsObjective: To explore a set of complete and feasible methods for the preparation of human umbilical cord mesenchymal stem cells and to lay a foundation for further experiments.Methods: The umbilical cord of newborn was extracted and digested by mechanical method.The surface markers of the cells were detected by flow cytometry.Results: A large amount of hUCMSCs could be obtained by adherent culture.The cells were long fusiform or polygonal,vortex or income like distribution.After 8th passage,the cells were still active and proliferative.Flow cytometry showed that after the 3rd passage,Expression levels of cell surface markers CD90,CD44,CD105,CD73 were all above 90% while the expression levels of CD34,CD43,CD19,CD11 b and HLA-DR were very low at passages 1-9.In accordance with the biological characteristics of MSCs.Conclusion: The adherent growth of hUCMSCs can be obtained from umbilical cord warton glue by mechanical method.After culture and subculture,a large number of hUCMSCs can be purified and amplified to meet the needs of subsequent experiments.Part ?: Experimental study on repairing radiation-induced skin ulcer by human umbilical Cord Mesenchymal Stem cells in Rats.Objective: To investigate the therapeutic effect of hUCMSCs transplantation on radiation induced skin ulcer in rats.Methods: Sixty SD rats were randomly divided into 3 groups: sham group,radiation skin ulcer group and irradiated+hUCMSCs group.The animal model of radiation skin ulcer was established by irradiation of 4 Mev electron beam from linear accelerator to the skin of the buttocks of rats at a total dose of 45 Gy.After transplantation of human umbilical cord mesenchymal stem cells 3d.7d.14 d,the homing and proliferation of stem cells were observed.The healing rate of wound was observed and compared at 1 w,2 w,3 w,4w after transplantation.The pathological changes of skin tissue were observed by HE staining and Masson staining.Results: GFP labeled stem cells were observed and proliferated in the wound tissue 3d.7d.14 d after human umbilical cord mesenchymal stem cell transplantation.The wound healing rate of h CUMSCs group was significantly higher than that of radiation skin ulcer group(P < 0.05).Pathological observation showed that there were more skin adnexal hyperplasia such as reepithelialization,collagen fiber proliferation and sebaceous gland of sweat gland in the treatment group of hUCMSCs than those in the group of radiation skin ulcer.Conclusion: Human umbilical cord mesenchymal stem cells can successfully homing,proliferating and accelerating wound healing and improving the quality of skin repair in radiation skin ulcer tissue.Part ?: Proteomic study on repair of radiation skin ulcer by human umbilical cord mesenchymal stem cells based on iTRAQ techniqueObjective: To search for differential proteins of human umbilical cord mesenchymal stem cells(h UMSCs)in repairing radiation induced skin ulcer in rats by proteomics and to find the signal pathway involved in repair.Methods: LC-MS / MS was combined with iTRAQ technique.The false injury group,radiation skin ulcer group and human umbilical cord mesenchymal stem cell treatment group were marked 114,115 and 116 respectively.After mixed samples were identified by mass spectrometry,the protein with a differential multiple of more than 1.2(down-regulated)and P-value less than 0.05 were regarded as differentially expressed proteins.Results: 540 differentially expressed proteins were screened in radiation skin ulcer group compared with sham injury group,including 233 up-regulated and 307 down-regulated.A total of 568 differentially expressed proteins were screened in the human umbilical cord mesenchymal stem cell treatment group compared with those in the radiation skin ulcer group,of which 339 were up-regulated and 229 were down-regulated.Differential proteins include keratin and proteins related to cell apoptosis and cell proliferation.The enrichment analysis of differentially expressed protein KEGG pathway showed that p53 cell signaling pathway,PI3K-Akt cell signal pathway,MAPK cell signaling pathway and Wnt signaling pathway are involved in the repair of radiation skin ulcer by human umbilical cord mesenchymal stem cells.Conclusion: this study shows that iTRAQ-LC-MS/MS can be used to screen differentially expressed proteins quickly and effectively.This will lay a foundation for the study of the repair mechanism of chronic skin wounds by h CUMSCs transplantation.Part ?: Study on the mechanism of human umbilical cord mesenchymal stem cells repairing radiation-induced skin ulcersObjective: To investigate the mechanism of hUCMSCs transplantation in repairing chronic refractory wounds by Western blot or immunohistochemistry.Methods: The expression of keratin 19 was detected by immunohistochemistry.Western blot method was used to detect caspase 3 and TUNEL staining to observe the change of apoptosis in wound tissue.Western blot method was used to detect the proliferation of wound tissue cells.Results: The expression of keratin 19 was decreased in radiation-induced skin ulcer and increased at 1 and 4 weeks after hUCMSCs transplantation(P < 0.05).TUNEL staining and Western blot detection of caspase 3 showed that electron beam irradiation increased the apoptosis of wound tissue.hUCMSCs transplantation can reduce the apoptosis of wound tissue.Western blot assay was used to detect PCNA.It was found that electron beam irradiation inhibited the proliferation of wound tissue cells and hUCMSCs could increase cell proliferation.Conclusion: hUCMSCs can promote the growth and proliferation of skin basal stem cells through paracrine effect and inhibit apoptosis which may be the mechanism of hUCMSCs transplantation to repair chronic skin wounds.