The Effect And Mechanism Study Of Menstrual Blood-derived Endometrial Stem Cells On Improving The Diabetic Foot Ulcer

BackgroundDiabetic foot ulcer(DFU)is a serious complication of diabetes mellitus,50% of patients with ulcers are infected,increasing the risk of lower limb amputation,and 69% of patients with diabetic amputation can not survive for more than five years.Despite the development of new treatments for DFU,recurrent infections and ulcers are difficult to improve until now,and seriously affect the physical and mental health of patients.In recent years,stem cell-based therapies have been considered as effective treatment for DFU.Recently,menstrual blood-derived endometrial stem cells(MenSCs)have gradually become a promising alternative for stem cell-based therapy due to their comprehensive advantages,which include their ability to be periodically and non-invasively collected,their abundant source material,their stable donation,their superior proliferative capacity and their ability to be used for autologous transplantation,and have shown positive therapeutic potential for the treatment of various diseases.Objective1.Effects of MenSCs on angiogenesis and granulation in vitro and in vivo.2.To investigate the therapeutic effect of MenSCs on diabetes mellitus and wound healing.Method1.To examine the expression of angiogenic factors in the conditioned medium of MenSCs(MenSC-CM)by protein array;The effects of MenSC-CM on the migration and adhesion of HUVECs were examined by Transwell assay and adhesion experiments,respectively.The effect of MenSCs-CM medium on promoting angiogenesis was examined by tube formation assay in vitro;Matrigel plug test was used to examine the angiogenesis potential of MenSCs in vivo.2.The effects of MenSC-CM on the proliferation,migration and adhesion of skin fibroblasts(BJ cells)were examined by MTT assay,Transwell assay and adhesion experiments.The non-contacted co-culture of MenSCs and BJ cells was used to detect the effect of MenSCs on the production of reactive oxygen species(ROS)in BJ cells in the presence of high glucose and LPS.3.The diabetic foot ulcer rat model was constructed,and the fasting blood glucose and wound healing rate of the model rat were examined at the time point of 1d,3d,5d,7d and 9d after MenSCs transplantation;The glucose tolerance of experimental rats were detected at 9 days;The pancreas was conventionally sectioned and performed with HE and Ki67 staining;The liver was conventionally sectioned and stained with PAS.Result1.The results of protein array showed that MenSCs can secrete a variety of angiogenic factors;subsequently,MenSC-CM can enhance the migration and adhesion ability of HUVECs;furthermore,In tube formation test,MenSCs-CM treated HUVECs can form more nodes,junction and branches,and the total length and branch length of tubes significantly increased;The result of matrigel plug test showed that MenSCs can significantly promote the increase of vascular density.2.The results of MTT assay,Transwell assay and adhesion experiment showed that MenSCs could significantly promote the proliferation,migration and adhesion of BJ cells;and the production of ROS in BJ cells was significantly reduced in the non-contacted co-cultured system with MenSCs.3.The diabetic foot ulcer rat model was successfully constructed;since 5d after the MenSCs transplantation,the fasting blood glucose in experimental rats began to decrease in the MenSCs treated rats,and the capacity of glucose tolerance in the MenSCs treated rats increased significantly at 9 days;The results of HE and Ki67 staining also showed that the islets of the MenSCs treated rats recovered significantly,and the glycogen synthesis and storage capacity in the liver of the MenSCs treated rats increased significantly;The wound healing rate of the MenSCs treated rats was significantly improved,and the size of wound in the foot of MenSCs treated rats was about 35% smaller than that of the control rats.Conclusion1.The in vitro and in vivo results showed that MenSCs can promote the angiogenesis and granulation.2.MenSCs transplantation can systemically improve diabetic symptoms and promote wound healing of diabetic foot ulcer.