Preliminary Study On The Proliferation And Differentiation Of HaCaT Promoted By GMSCs Through Low Expression Of Macrophage Movement Inhibitory Factor

Objective: Implant-supported prostheses is becoming the first choice to repaire missing teeth.Adequate amount of keratinized gingival can improve the long-term stability of the implant and maintain the oral health.Gingiva-derived Mesenchymal Stem Cells(GMSCs)are adult stem cells extracted from gingival tissues with the characteristics of mesenchymal stem cells,which have good proliferation and differentiation abilities and can secrete a variety of cytokines and play an important role in the process of tissue damage repair.Therefore,studying the role of GMSCs in gingival keratinization during the repair process is important for further understanding the differentiation process of keratinocytes,and it is also clinically relevant for guiding the development of drugs and technologies related to pro-keratinization.In this experiment,we investigated the effect of GMSCs on the differentiation of immortal human skin keratinocytes(HaCaT)by detecting the relevant cytokines in the supernatant of GMSCs.Methods: In this study,primary human GMSCs were isolated from human gingival tissue and characterized by surface antigen analysis.By culturing human primary GMSCs and human foreskin fibroblast(HFF-1),collecting the cell supernatants and culturing HaCaT with the supernatant for 24 hours,the change of proliferation and migration ability of HaCaT was detected by Cell Counting Kit-8(CCK8)assay,scratch assay,and Western Blot(WB)to analyze the changes in protein levels of keratin.One of the cytokines with large differences,macrophage migration inhibitory factor(MIF),was selected as the target of the study.Different concentrations of MIF were tested for its effect on the cytotoxicity of HaCaT and the effect on HaCaT proliferation and differentiation were also investigated.After silencing MIF with small interfering RNA(si RNA)in HFF-1 cells with high MIF expression,the effect of the cell supernatant and GMSCs cell supernatant on HaCaT proliferation and differentiation was investigated.Finally,by establishing a palatal gingival defect model in C57BL/6 mice,phosphate buffered saline(PBS)and MIF were injected around the defect respectively,and the palatal samples from experimental and control mice were collected during the process of damage repair(day 5 after the operation)and analyzed by appearance images.Hematoxylin-eosin(H&E)staining and immunohistochemistry(IHC)staining were also used to observe the repair of gingival soft tissues and epithelium in the palate of mice.All the differences were statistically analyzed.Results: Compared to HFF-1,cell supernatants from GMSCs were able to increase HaCaT migration and proliferation capacity and more mature differentiation.After analyzing the cell supernatants of the two different cells using cytokine arrays,the differentially expressed cytokine MIF was screened among them and the low expression of MIF in GMSCs,both at the protein level and at the gene level,was determined.Stimulation of HaCaT using human recombinant MIF protein had little effect on HaCaT proliferation,but inhibited HaCaT differentiation at high concentrations,and this inhibition was rescued after using HFF-1 cell supernatant with knockdown of MIF.The results of in vivo experiments showed that the healing process of gingival defects on the palate of mice was inhibited after local injection of MIF,while epithelial differentiation at the defect site was suppressed.Conclusion: Cell supernatant from GMSCs promotes the proliferation and differentiation of HaCaT compared to HFF-1,correlating with the low expression of MIF in GMSCs;low concentration of MIF recombinant protein enhances keratin expression and promotes gingival tissue healing in the mouse palate.