Role Of MSCs Homing In The Process Of Skin Wound Healing And Skin Appendages Regeneration

ObjectiveMesenchymal stem cells(MSCs)play an important role in the skin and appendages repair and regeneration process after trauma.However,the mechanism of its role remains unclear.Thus,in order to provide experimental basis for the skin wound healing in both structure and function,investigation the effect of MSCs recruitment and differentiation,paracrine effect of homing MSCs during wound healing and the mechanism of reprogramming mesenchymal stem cells into sweat gland like cells are beneficial to guide the MSCs repairment and regeneration of multiple tissues at the same time.Methods1?The red fluorescence probe marked MSCs were infused in the blank group and the wound group.Immunofluorescence and flow cytometry were used to detect the homing of MSCs.Two days after total skin loss,MSCs were separated from bone marrow and peripheral blood.Flow cytometry was employed to detect the expression of CD29,CD44,CD90 and CD 105 to evaluate the stemness of MSCs and the expression of OPN,Nestin,Myosin and CK19 to verify the direction of differentiation.2?The main active molecules in the supernatant of MSCs culture medium(MSC-CM)were detected by microbead method.And flow cytometry was used to detect the cell activity,cell cycle and apoptosis of Human immortalized keratinocyte after treated with MSC-CM.The effect of epithelial-mesenchymal transition(EMT)-like progress of MSC-CM treated HaCaT cells was detected by Scratch test,RT-qPCR,Western blot and immunofluorescence.The effect of MSCs culture supernatant on healing speed in vivo was evaluated after establish a model of full layer skin defect in mice.3.Using CRISPR/dCas9-effector(dCas9-E)technology,the lentivirus containing single-guided RNA(sgRNA)targeting the EDA promoter sequence was used to infect MSCs.RT-qPCR,Western blot and the immunofluorescence were used to detect the expression of sweat gland cells related biomarkers and the ultrastructure was used by transmission electron microscopy.Western blot and immunofluorescence were used to evaluate the activity of sweat gland cells related signal molecules and the intracellular localization.Mouse paw scald model was used to evaluate perspiration function of MSCs treated with Cas9-E.Results1.The red fluorescent signal was shown by immunofluorescence in the tissue section of the wound,and the results of flow cytometry also confirmed that the number of labeled MSCs in the wound tissue was increased.At the same time,in the wound model of mice with different weeks of age,the markers(CD29,CD44,CD90,CD 105)of MSCs from both bone marrow and peripheral blood were changed little compared with control group.In the differentiation markers,the expression trend of OPN and Myosin was consistent with the control group,while Nestin and CK19 were significantly higher than those in the blank control group.2?Microbead experiments confirmed that TGF-? is the main active molecule in the supernatant of MSCs culture medium.MSC-CM can promote the proliferation and migration of HaCaT cells.Furthermore,RT-qPCR,Western blot and immunofluorescence results showed that MSCs culture supernatant can induce EMT-like changes in HaCaT cells,while TGF-P specific inhibitor SB-431542 can reverse this changes.The animal experiment showed that the skin wound can accelerate healing after treated with MSC-CM.3.We have successful predicted of EDA gene promoter sequence and constructed a lentivirus containing sgRNA sequences targeting the EDA promoter.The screened positive cell lines(dCas9-E)were confirmed that CEA,CK19 and other sweat gland markers were positive by RT-qPCR,Western blot and immunofluorescence test and transmission electron microscopy showed that it contained a villous structure.RT-qPCR,Western blot and immunofluorescence results showed NF-?B could be avtivated by expression of EDA,and then the activated-NF-?B could entered into nucleus to promote the expression of Shh and Cyclin D1 in the MSCs.ConclusionAfter skin trauma,the wound tissue can recruit MSCs homing through the peripheral circulation,and can induce the initial differentiation of MSCs into the direction of nerve forming and skin formation.The homed-MSCs from wound tissue could regulate the local microenvironment through paracrine effect and the secreted active molecule TGF-? could accelerate the EMT process of epidermal cells through TGF-?/Samd signaling pathway,thereby accelerating wound healing.In the meaning time,using CRISPR/Cas9 could reprogram MSCs into sweat gland-like cells,solving the problem of local sweat gland regeneration after wound healing.Thus,these could provide a theoretical basis for study the synchronous regeneration of multi tissue after trauma.