| Objectives:Vitro experiments were conducted to investigate the proliferation of umbilical cord mesenchymal stem cells after improving the culture environment of umbilical cord mesenchymal stem cells,such as coculture with microdermis.Animal experiments were conducted to explore the effects and mechanisms of improving the microenvironment of umbilical cord mesenchymal stem cells transplantation on wound healing in rats,and to provide theoretical basis for the future clinical application of umbilical cord mesenchymal stem cells.Methods:1.In vitro cell experiments,UC MSCs were co-cultured with microdermis in vitro.The first group was only UC MSCs,the second group was 1 cm2 neonatal SD rat microdermis+UC MSCs.The morphological changes of UC MSCs were observed.The proliferation of umbilical cord mesenchymal stem cells was detected by CCK8 method.Then the serum of severely burned rats was used to simulate the microenvironment of burn.UC MSCs,microdermis and rhEGF were co-cultured as following groups:group ?:10%burn serum+UC MSCs;group ?:10%burn serum+1 cm2 neonatal SD rat microdermis+UC MSCs;group ?:10%burn serum+20ng/mL rhEGF+UC MSCs;group ?:10%burn serum+20ng/mL rhEGF+1 cm2 neonatal SD rat microdermis+UC MSCs.The interaction between UC MSCs,microdermis and rhEGF were observed.The morphological changes of mixed cultured cells were observed.The proliferation of umbilical cord mesenchymal stem cells was detected by CCK8.2.Animal experiments were conducted to establish a rat model of deep second-degree to third-degree scald.Escharectomy was performed 3 days after scalding.The rats were treated according to the following two groups:UC-MSCs+allogenic reticular skin group and UC-MSCs+ rhEGF+ autogenous microskin+allogenic reticular skin group.After treatment,routine bandaging and fixation were performed.The wound healing was observed by HE staining.The infiltration of neutrophils and macrophages was detected by MPO and MAC-2 immunohistochemical staining.The expression of pro-inflammatory factors IL-1?,IL-6,TNF-?,anti-inflammatory factors IL-4,IL-10,chemokines SDF-1 and CXCR 4 were detected by EILSA kit.Results:1.After co-culture of UC MSCs with microdermis in vitro,the number of UC MSCs cells first decreased and then increased,which was basically the same as that of UC MSCs alone at 14 days.In the microenvironment simulated by 10%burn serum,the cell viability of 10%burn serum+UC MSCs group was higher than that of the other three groups at 3 and 7 days.The cell viability of 10%burn serum+microdermis+UC MSCs group and 10%burn serum+rhEGF+microdermis+UC MSCs group were significantly increased at 14 days,and there was no difference between the two groups.2.The wound healing of the second group was better than that of the first group.From the 3rd day after operation,the epidermis structure was gradually intact,the dermis was gradually formed,the inflammatory reaction was gradually alleviated,the fibroblasts were gradually enlarged,the collagen fibers and capillaries were gradually formed and increased,and the structure of hair follicles was gradually formed.The hair follicle structure and the number of new blood vessels in the second group were more than those in the first group,and the healing condition was better.The expression of MPO in the wounds of the second group rats was lower than that in the first group on the 3rd,14th and 28th day after operation.The difference was statistically significant on the 14th and 28th day after operation(P<0.05).There was no significant difference on the 3rd day after operation(P>0.05).The expression of MPO in the wounds of rats in the second group was higher than that in the first group on the 7th day after operation,but there was no significant difference(P>0.05).On the 3rd,7th,14th and 28th day after operation,The expression of MAC-2 in the wounds of the second group rats were lower than those in the first group.There was significant difference between 7th days and 28th days after operation(P<0.05),but there was no significant difference between 3rd days and 14th days after operation(P>0.05).The expression of IL-1beta in the first group was higher than that in the second group on the 14th day after operation in ELISA test.There was significant difference between the two groups(P<0.05).The expression of TNF-alpha in the first group was lower than that in the second group on the 7th day after operation.There was significant difference between the two groups(P<0.05).The expression of SDF-1 in the first group was higher than that in the second group on the 14th day after operation.There was significant difference between the two groups(P<0.05).There was no significant difference in other groups(P>0.05).Conclusions:1.The co-culture of UC MSCs and microdermis in vitro does not affect the proliferation of UC MSCs,so it is feasible to co-use them.In the microenvironment simulated by 10%burn serum,10%burn serum+microdermis+UC MSCs group and 10%burn serum+rhEGF+microdermis+UC MSCs group decreased cell viability at 3 and 7 days of co-culture,and increased cell viability at 14 days of co-culture.2.Compared with UC-MSCs+allogenic reticular dermis group,UC-MSCs+rhEGF+autologous microdermis+allogenic reticular dermis group has obvious promoting effect on wound healing in rats.Compared with UC-MSCs+allogenic reticular dermis group,UC-MSCs+rhEGF+autologous microdermis+allogenic reticular dermis group significantly reduced neutrophil infiltration and macrophage infiltration in burn wounds 3rd,7th,14th and 28th days after UC-MSCs transplantation.There was no significant difference in the expression of pro-inflammatory,anti-inflammatory and chemokines between UC-MSCs+rhEGF + autologous microdermis+allogenic reticular dermis group and UC-MSCs+allogenic reticular dermis group. |
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