Experimental Study On Differentiation Of HADSCs Into Sweat-like Cells

Objective: The purpose of this study was to explore a new way to induce sweat adenoid cells in vitro by transfecting human adipose-derived stem cells(ADSCs)with lentiviral vectors overexpressing ectodermal hypoplasia gene(EDA)and its receptor gene(EDAR),and to further understand the key genes(EDA and its receptor EDAR)that can regulate the differentiation of hADSCs into sweat adenoid cells,further demonstrating that hADSCs can become better seed cells for the induction and differentiation of sweat adenoid cells,providing high quality sweat adenoid cells for subsequent composite sheep acellular dermal matrix(Acellular Dermal Matrix ADM)to promote wound healing experiments and sweat gland regeneration therapy,and bringing hope for clinical large area burns to promote wound healing and sweat gland regeneration.Methods: Discarded adipose tissue after surgery was digested using 0.1% collagenase type I,and the cells isolated in vitro were subcultured.The fourth generation of cells was selected to observe the cell morphology,detect the expression of cell markers CD34,CD44,CD90,and CD105 by immunofluorescence,and detect the adipogenic,chondrogenic,and osteoblastic differentiation ability to identify whether the obtained cells were hADSCs.The hADSCs obtained by transfection with lentivirus HBLV-Zs Green-PURO carrying EDA and EDAR and unloaded lentivirus,respectively,were named EDA-hADSCs group,EDAR-hADSCs group,GFP-hADSCs group(transfected with unloaded lentivirus)and hADSCs group(not transfected)according to different transfection genes,and the above 4 cells were cultured under the same conditions.The expression of green fluorescent protein(GFP)in each group of cells was observed by fluorescence microscopy and photographed,and the expression levels of m RNA and protein of EDA and EDAR in each group of cells were detected by RT-qPCR and Western blot to identify whether the transfection was successful;the expression levels of CEA,CK8,and CK18 m RNA and protein in each group of cells were detected by RT-qPCR and Western blot to identify whether hADSCs differentiated sweat adenoid cells after transfection.Results: 1.hADSCs were successfully isolated and extracted from human adipose tissue in this experiment.Immunofluorescence staining revealed that hADSCs positively expressed CD44,CD90,and CD105,but not CD34,and the isolated hADSCs had the ability to differentiate into adipocytes,osteocytes,and chondrocytes.2.stable cells were observed under a fluorescence microscope,and green fluorescence was observed suggesting successful transfection.RT-qPCR showed that the expression of EDA and EDAR m RNA was significantly up-regulated in stable cells,suggesting that they possessed higher transcript levels,which were significantly different compared with the overexpression control group as well as the blank control group(P < 0.01).Western blot results showed that EDA and EDAR proteins were significantly expressed in the target gene transfection,but not in the overexpression control group and the blank control.3.RT-qPCR results showed that the expression of CEA,CK8,and CK18 m RNA was significantly up-regulated in the target genome 1 and target genome 2,which were significantly different from the overexpression control group as well as the blank control group(P < 0.01).Westernbolt results showed that CEA,CK8 and CK18 proteins were significantly expressed in target genome 1 and target genome 2,but not in overexpression control group and blank control group.Conclusion: This experiment preliminarily explored the effect of overexpression of EDA and EDAR genes on the differentiation of hADSCs into sweat adenoid cells,confirmed that overexpression of EDA or EDAR genes can induce the differentiation of hADSCs into sweat adenoid cells,provided a basic theoretical basis for the induction and differentiation of hADSCs into sweat adenoid cells in vitro using many other types of cells,and brought hope for sweat gland regeneration therapy after sweat gland damage in patients with large area burns.Sweat gland regeneration therapy holds promise.