| Objectives:Burns are common traumas in peacetime and wartime,and the key to saving burn patients is to close the wound as early as possible.Recent studies have found that mesenchymal stem cells(MSCs)can effectively alleviate the inflammatory response of the wound and promote the proliferation and migration of wound fibroblasts.In this study,MSCs were isolated from human MSCs to promote wound healing To explore whether umbilical cord mesenchymal stem cells can promote the repair of burn wounds by regulating autophagy effect and to observe the effect of umbilical cord mesenchymal stem cells on local fibroblasts of burn wounds,and to explore its specific mechanism,so as to provide new ideas and experimental basis for the clinical treatment of severe burn wounds.Methods:1.hUC MSCs cell culture and identification:use tissue block adherence method to isolate and culture hUC MSCs.Observe the morphology and structure of cells with an inverted phase-contrast microscope;flow cytometry identifies the expression of cell surface markers CD34,CD45,CD90,CD 105 and Oct-4;oil red O staining and alizarin red staining methods identify cell adipogenesis and growth The differentiation potential of bone.2.The qualified cells were cultured in groups and treated with rapamycin at concentrations of 0.03μM,0.3pM,3μM and 3-Methyladenine(3-MA)at concentrations of 0.3nM,3nM,30nM After culturing on 1d,2d,3d,4d,5d,add CCK-8 to detect cell viability to obtain the best concentration and time.Then use the serum of severe burn patients to simulate the burn microenvironment to culture hUC MSCs.The experiment is divided into 10%normal serum+hUC MSCs group,10%burn serum+hUC MSCs group,10%burn serum+hUC MSCs+DMSO group,and 10%burn Serum+hUC MSCs+3-MA(0.3nM)group,10%burn serum+hUC MSCs+rapamycin(0.03μM)group.After culturing for 3 days,the expression of autophagy-related proteins Lc3Ⅱ/Ⅰ,p62,Beclinl was detected by WB;the exogenous GFP-LC3 fluorescent autophagy indicator system was used to mark and track the changes of LC3B;the CCK-8 method was used to detect the proliferation of hUC MSCs.3.hUC MSCs were co cultured with human dermal fibroblast(HDF)in Transwell cell co culture system to simulate burn microenvironment.The experimental groups were HDFa+10%normal serum(upper chamber)/10%normal serum(lower chamber)(simulated gravity),HDFa+burn serum(upper chamber)/burn serum(lower chamber),HDFa+burn serum(upper chamber)/hUC MSCs+burn serum(lower chamber),HDFa+burn serum(upper chamber)/hUC MSCs+burn serum+DMSO(lower chamber),HDFa+burn serum(upper chamber)/hUC MSCs+burn serum+rapamycin(lower),HDFa+burn serum(upper)/hUC MSCs+3-mA + burn serum(lower).After 3 days of culture,the expression of collagen type I(Col-I)and collagen type III(Col-3)was detected by immunofluorescence staining.The secretion levels of TGF-β1,PGE-2,IL-10,IDO and IL-6 and the expression of autophagy proteins lc3Ⅱ/Ⅰ and p62 in hUC MSCs were detected by ELISA.Results:1.hUC MSCs cell culture and identification:primary cells are obtained by tissue adherence method,and 70%-80%fusion is formed around the tissue block.The cells are polygonal,irregular,and large in size;when the cells reach the Pi generation,The cell morphology is still polygonal,and the cell volume is larger;after the cells are passed to the P2 generation,the cell morphology gradually becomes thinner and longer,mainly long spindle cells,and some are polygonal;P3 generation-P5 generation cells are present Long fusiform,vortex-like growth.When the P,generation cells are 80%-90%fused,flow cytometry shows that the cells highly express CD90 and CD 105,but do not express CD34 and CD45,and the cells positively express the molecular marker of pluripotent stem cells Oct-4.After hUCMSCs induced osteogenic culture for 28 days,calcium deposits were observed under the microscope,and the calcium deposits were stained with Alizarin Red to present dense red nodules.After hUC MSCs were cultured for 28 days after adipogenic induction,small and round fat particles were seen under the microscope,and oil red 0 stained orange-red.These results indicate that the cells obtained from the adherent culture of the tissue block conform to the characteristics of MSCs-specific molecular expression,and have the potential for osteogenic and adipogenic differentiation.2.WB was used to detect the marker protein of Akt/mTOR.The expression of autophagy related proteins lc3Ⅱ/Ⅰ,p62 and Beclin-1 were detected after 3 days of culture.The results showed that:the expression of lc3IⅡ/Ⅰ,p62 and Beclinl in 10%burn serum+hUC MSCs group was significantly higher than that in 10%normal serum +hUC MSCs group(P<0.01),suggesting that bum serum can stimulate the autophagy of hUC MSCs;the expression of lc3Ⅱ/Ⅰ,p62 and Beclin-1 in 10%burn serum+hUC MSCs+rapamycin group was significantly higher than that in 10%burn serum+hUC MSCs group The expression of lc3Ⅱ/Ⅰ and Beclin-1 in MSCs group increased(P<0.01),and the expression of p62 decreased(P<0.05),which indicated that rapamycin promoted autophagy of hUC MSCs in burn wound microenvironment;the expression of lc3ii/I and Beclinl in 10%burn serum+hUC MSCs+3-MA group was lower than that in 10%burn serum+hUC MSCs group(P<0.05),and the expression of p62 was up-regulated,but there was no statistical significance.WB test verified that burn serum can stimulate the autophagy of hUC MSCs,and rapamycin can further promote the autophagy of hUC MSCs in the burn wound microenvironment.3-MA inhibited the autophagy of hUC MSCs in the burn wound microenvironment In addition,CCK-8 assay showed that burn microenvironment could effectively stimulate the proliferation and survival of hUC MSCs,rapamycin could further promote the proliferation and survival of hUC MSCs in burn environment,while 3-MA inhibited hUC MSCs The proliferation and survival of MSCs in bum environment,DMSO dose did not produce toxic and side effects on hUC MSCs.3.1n the Transwell cell co-culture system,collagen Ⅰ,Collagen Ⅲimmunofluorescence and DAPI staining were performed by immunofluorescence cytochemical staining.The results showed that hUC MSCs transplanted in the bum microenvironment can make the type Ⅰ collagen of wound fibroblasts.Increased expression of type Ⅰ,Col-1)and type Ⅲ collagen(collagen type Ⅲ,Col-3),and at the same time promotes the autophagy level of hUC MSCs in the burn environment,which can further increase the Col-1 and Col-1 of wound fibroblasts.The expression of 3,on the contrary,decreases.The secretion levels of TGF-p1,PEG-2,IL-10,IDO and IL-6 in MSCs,as well as the expression of MSC autophagy-related proteins LC3Ⅱ/Ⅰ and p62 were detected by ELISA.The results showed that the secretion of TGF-β1 was positively correlated with the autophagy-related protein of hUC MSCs LC3Ⅱ/Ⅰ,and the secretion of PGE-2 was positively correlated with the autophagy-related protein p62 of hUC MSCs.Conclusions:1.The cells obtained by the tissue block adherence method conform to the specific molecular expression characteristics of hUC MSCs,and have multiple differentiation potentials.2.The burn environment can stimulate the autophagy effect of hUC MSCs,which enhances the proliferation and survival ability of mesenchymal stem cells;and the autophagy effect of activating hUC MSCs can further enhance the proliferation and survival ability of mesenchymal stem cells in the microenvironment of burn wounds.3.Activation of autophagy of hUC MSCs can promote the proliferation and migration of burn wound fibroblasts,and autophagy may promote the proliferation and migration of burn wound fibroblasts by inducing mesenchymal stem cells to paracrine TGF-β1 and inhibiting wound inflammation. |
