Experimental Study On Promoting Wound Healing By HADSCs Of Overexpressing GH Combined With Particulate Sheep Acellular Dermal Matrix

Objective To construct growth hormone-human adipose-derived stem cells(GH-hADSCs)and investigate the effects of GH-hADSCs on proliferation and migration of fibroblasts.Sheep acellular dermal matrix(SADM),particulate sheep acellular dermal matrix(pSADM)and silicone membrane were prepared.It was combined with GH-hADSCs to form tissue engineered skin.Methods(1)hADSCs were isolated by enzyme digestion.Adipogenic,chondroblast and osteoblast differentiation and immunofluorescence staining of mesenchymal stem cells were used to identify the cells as hADSCs.(2)Construct GH-overexpressing lentivirus,divide hADSCs into GH group,empty group,and control group.The above three groups were transfected with GH-overexpressing lentivirus,empty lentivirus,and no infection.(3)The mRNA and protein expressions of GH and IGF-1 and the expression of ERK1/2 pathway in cells and supernatants of each group were detected by RT-qPCR,Western blot and ELISA.(4)The effects of GH group and empty group on the migration and proliferation of fibroblasts were detected by the scratch test,Transwell test,and MTS test.Detect the expression of related genes in fibroblasts after co-culture with GH-ADSC by RT-qPCR.(5)After inhibiting GH-hADSCs with ERK inhibitor SCH772984,the expression changes of related genes in fibroblasts were observed after co-culture with GH cells.(6)Sheep-acellular dermal matrix was prepared by trypsin digestion and Triton X-100 method.The cytotoxicity and acellular efficiency of SADM by the two methods were detected.Part of SADM was prepared by microdermal machine to make pSADM.(7)GFP-hADSCs or hADSCs were inoculated on the surface of SADM and pSADM,and the morphology and number of cells on the scaffold were observed.(8)Liquid silicone was used to prepare silicone membrane and MTS assay was used to detect its cytotoxicity.Results(1)The hADSCs were isolated and identified,and cell lines of GH-hADSCs were successfully constructed.(2)The mRNA and protein expressions of GH in GH-hADSCs were significantly higher than those in no-load group and control group(P< 0.01),and the concentration of GH in supernatant was higher.The expression of ERK1/2 and p-ERK1/2 protein in GH-hADSCs was up-regulated.(3)GH-hADSCs promoted the migration and proliferation of fibroblasts better than GFP-hADSCs(P <0.05).(4)After GH-hADSCs co-cultured with fibroblasts,the expression of MMP-2gene was down-regulated,and the expression of type Ⅰ and Ⅲ collagen gene was up-regulated.(5)After inhibition of ERK protein in GH-hADSCs,the mRNA expression differences of these genes disappeared.(6)SADM and pSADM were successfully prepared,and GH-hADSCs were inoculated on their surfaces.It was observed by scanning electron microscope,HE staining and inverted fluorescence microscope that the cells grew well on the scaffold.(7)The silicone membrane was prepared and no obvious cytotoxicity was found by MTS assay.Conclusion In this study,lentivirus transfection was used to construct GH-hADSCs that can continuously secret GH and promote the biological function of fibroblasts.This process may be related to upregulation of intracellular ERK1/2 pathway.SADM is a tissue engineering scaffold material with excellent spatial structure and biocompatibility,on which hADSCs can be well multiplied,and the cell load can be further increased after making pSADM.The tissue-engineered skin prepared in this study may solve the current problem of recombinant human growth hormone in wound treatment,and provide a new idea for the study and application of tissue engineering in wound repair.