The Experimental Study On Substance P Combined With Epidermal Stem Cells To Promote Wound Healing And Nerve Regeneration In Diabetes Mellitus

Object:To explore diabetes mellitus rats skin epidermal stem cells methods of separation and culture in vitro and its biological characteristics, observe substance P combined with epidermal stem cells for the effects of diabetic diabetes mellitus rats wound healing and nerve regeneration,this study provides a new techology methods for diabetes mellitus rats wound repairing, establishes furture experimental basis and theoretical basis for clinic application.Methods:1) SD rats are randomly divided into diabetes mellitus group and normal control group. Diabetes mellitus rat model was made by intraperitoneal injection of streptozocin, and the normal control group was not treated. Full-thickness skins of the back of diabetes mellitus group rats and normal rats are taken respectively. Enzyme digestion combined with type IV collagen attachment method is used to separate, culture and choose rat ESCs. Morphologic change and cell colony formation are observed under inverted phase contrast microscope. Cell growth curve is detected by cell counting and cell colony formation rates are measured. The positive expressions of K19,β1-integrin are identified by immunocytochemical stain and picture analysis software. The integral absorbance value of positive cells are detected.2) Taking 48 SD rats successful models,causing the diameter of 1.8cm full-thickness dermal wounds by a special punch in the back on both sides of the spine. four wounds of each rat were randomly divided into four groups, group A:epidermal stem cells and substance P;group B:epidermal stem cells;goup C:substance P;group D:simple amniotic membrane control group. mong them group A and B individually use amnion membrane load epidermal stem cells transplantation, group A and C individually are injected with 250μl substance P,10-1mmol/L,around wound and the middle of the wound after transplation, In the same condition group B and group D which inject substance P solvent are taken as control, two times a day for 4 consecutive days.Dynamic observation for wound healing conditions in each group; observing histological wound tissue changes by HE staining,observing wound collagen changes and wound nerve regeneration conditions by immunohistochemistry collagenⅠ,Ⅲ, PGP9.5, and substance P staining.Results:1)The number of primary anchorage-dependent ESCs in diabetes mellitus group is decreased, the colony forming efficiency of ESCs in diabetes mellitus group(7.43%±1.21%) is significantly lower than that in the normal control group (15.37%±2.03%), two groups have difference statistical significance (P<0.01). ESCs of both groups expresses K19 andβ1-integrin. The integral absorbance values of positive cells for K19 andβ1-integrin in ESCs of diabetes mellitus group are respectively 86.17±10.54,26.35±7.63,are significantly lower than those in the normal control group 160.31±36.52,59.46±14.67,two groups also have difference statistical significance (P<0.01).2) Compared with group B,C and D, the wound healing time in group A is obviously short, the content of collagen I and III in skin tissues are obviously high.wound tissues in group A and C have a large number of PGP9.5 and substance P positive expression of nerve fiber regeneration, part of the nerve fiber terminals extending to the skin are close to the epidermis. The deeper wound tissues in group B and D only have a little of PGP9.5 and substance P positive expression of nerve fiber regeneration, there is no nerve fiber terminals extending to the epidermis.Conclusion:1) in this culture system conditions,we can successful separation and culture diabetes mellitus ESCs.The multiplication capacity in vitro rat ESCs in diabetes mellitus group is slower than normal skin, and it may be one of important factors that diabetes mellitus wound is hard to heal.2)Substance P combined with epidermal stem cells can effectively promote wound healing and nerve regeneration in diabetes mellitus, this study provides a new techology methods for diabetes wound repairing, establishes furture experimental basis and theoretical basis for clinic application.