| Objective:To establish diabetic rat model,and to isolate and culture the rat epidermal stem cells,The differential expression and correlation of IncRNA and mRNA of skin epidermal stem cells of diabetic rats and normal rats were investigated by gene chip and bioinformatics analysis methods,and their potential biological functions will be analyzed to provide new ideas and theoretical basis for diabetic wound healing.Methods:Ten healthy male SD rats were randomly divided into DM group and normal control group,5 rats in each group.Rats in the DM group were induced by intraperitoneal injected with Streptozocin(STZ,65 mg/kg)at one time to prepare diabetic rat model,while rats in the normal control group were injected with the corresponding volume of sodium citrate buffer.Epidermal stem cells were isolated and cultured in vitro from full-thickness skin samples of DM and normal rats.The growth of the cells was observed under inverted phase contrast microscope,and the positive expression of K19 and β1-Integrin was identified by immunocytochemical staining.Trizol was used to extract total RNA from 2 groups of cells,and the samples were amplified by the random primer method and transcribed into fluorescent cRNA.The concentration and activity of purified and labeled cRNAs were detected by NanoDrop ND-1000.The chip was hybridized,washed,fixed and scanned.Using software to extract the original data and screening the differentially expressed LncRNA and mRNA.GO and KEGG pathways were used to analyze the differentially expressed genes.Finally,LncRNA subgroup analysis was performed.Results:The level of random blood glucose of DM group was above 16.7mmol/L after four consecutive weeks later,and classic symptoms of diabetes was appeared.The cells isolated and cultured by enzyme digestion combined with rapid adhesion of typeⅣ collagen showed clonal growth,but the number of ESCS in DM rats was reduced compared with normal control rats,and the cell growth rate was slowed down.Immunocytochemical staining showed positive expression of CK19 and β1 integrin in both groups.The results were consistented with the characteristics of epidermal stem cells.A total of 121 LncRNA and 533 mRNA were expressed differentially by gene chip technology,including 39 LncRNA significantly up-regulated and 82 LncRNA significantly down-regulated,243 mRNA significantly up-regulated,and 290 mRNA significantly down-regulated.GO analysis showed that the differentially expressed mRNA were mainly involved in stress response,lipid response,reaction to oxygen-containing compounds,cell movement,cell migration and other biological processes;Molecular functions showed that the up-regulated or down-regulated mRNA mainly had the functions of Oxidoreductase activity,signaling receptor binding,GTP binding,monooxygenase activity,heme binding,transferase activity,protein kinase activity,signaling receptor activator activity and so on.KEGG enrichment analysis suggested that the differential genes were related to fatty acid metabolism,PPAR signaling pathway,chemokine signaling pathway,P53 signaling pathway,Apelin signaling pathway,PI3K-Akt signaling pathway,etc.Finally,there were 2 differentially expressed antisense LncRNA and 7 differentially expressed LincRNA and their adjacent differential coding genes were screened by subgroup analysis.Conclusions:(1)Epidermal stem cells from diabetic rats and normal rats were isolated and cultured in vitro after established the diabetic rat model,then differential expression lncRNA and mRNA were further screened out.(2)GO analysis and pathway analysis showed that differentially expressed genes were involved in a variety of biological processes,which may be related to oxidative stress injury of diabetic epidermal stem cells,resulting in the change of cell proliferation,differentiation ability,and apoptosis and senescence.(3)In this study,differentially expressed lncRNA were screened out from diabetic rat epidermal stem cells,and then subgroup analysis screened out differentially expressed LncRNA and their adjacent differentially coding genes.LncRNA may become the potential biological target in diabetic wound healing. |
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