Effect Of Wnt/?-catenin Signaling Pathway And P38 Signaling Pathway On The Maintenance Of Stemness Of Human Stem Cells From The Apical Papilla By BFGF

Objective: After blocking Wnt/?-catenin signaling pathway and p38 signaling pathway of human stem cells from the apical papilla(SCAP)in vitro On the basis of previous experiments,the effects of basic fibroblast growth factor(bFGF)on proliferation,osteogenic differentiation and stemness gene expression of stem cells were observed.To explore the role of Wnt/?-catenin signaling pathway and p38 signaling pathway in the maintenance of SCAP stemness by bFGF,and to explore whether there is interaction between the two signaling pathways.Methods:(1)SCAP were primarily cultured and isolated from human third molar papilla by enzyme digestion method and limited dilution technique.(2)Immunofluorescence staining: The expression of STRO-1,CD24,vimentin and keratin in SCAP mesenchymal stem cells were detected.(3)Alizarin red and oil red O staining: After 4 weeks of induction culture with osteogenic adipogenic induction medium,the ability of multidirectional differentiation of SCAP was identified by alizarin red and oil red O staining.(4)The experiment was divided into eight groups: experimental group1: bFGF+DKK-1,experimental group 2: DKK-1,experimental group 3: bFGF+SB203580,experimental group 4: SB203580,experimental group 5: bFGF+DKK-1+SB203580,experimental group6: DKK-1+SB203580,positive control group: bFGF group,negative control group:?-MEM group.CCK-8 was used to detect cell proliferation: DKK-1 was used to block Wnt/?-catenin signaling pathway,SB203850 was used to block p38 signaling pathway,two signaling pathways were blocked,and CCK-8 was used to detect the proliferation of SCAP cells treated with 20 ng/mLbFGF at 1,3,5 and 7 days.(5)EdU fluorescence microscopy kit to detect new cells: Use method(4)Grouped with the same blocker treatment,EdU labeled each group within 24 hours of the fourth day of treatment.(6)Alkaline phosphatase(ALP)test box staining and alizarin red staining: DKK-1 was used to block Wnt/?-catenin signaling pathway,SB203850 was used to block p38 signaling pathway,and two signaling pathways were blocked respectively.After 14 days of inductionculture with 20 ng/mLbFGF osteogenic induction solution,SCAP cells in each group were stained with ALP and alizarin red.(7)qPCR was used to detect the transcription levels of osteogenic marker genes DSPP,ALP,OCN,BSP,OSX and Runx2.Use method(4)Grouped with the same blocker treatment SCAP cells were cultured in the same blocker medium containing 20 ng/mL bFGF four days later,and then the transcription of stemness genes Nanog,Oct4,Sox2 and Rex1 was detected by qPCR.Results:(1)Primary SCAP cells were obtained by enzymatic digestion in vitro,and colony adherent growth was observed under the microscope about a week.The cells were short fusiform,rich in cytoplasm and small inclusions.When the cells grew and fused to about80%,SCAP cells were cloned and screened by limited dilution method,and then expanded and cultured.The cell morphology was more consistent and the growth rate was faster.(2)Immunofluorescence staining showed that STRO-1,CD24 and vimentin antibody staining were positive in cytoplasm,keratin antibody staining was negative in cytoplasm,and the nuclei were normal DAPI staining.(3)Identification of multidirectional differentiation ability of cells: Alizarin red/oil red O staining was performed at 4 weeks after osteogenesis and adipogenic induction,and mineralized nodules and lipid droplets were observed.(4)CCK-8 was used to detect the cell proliferation at 1,3,5 and 7 days.At 3,5 and 7 days,the cell activity of positive control group was higher than that of negative control group.The cell activity of bFGF+DKK-1 group and bFGF+SB203580 group was lower than that of positive control group.The cell activity of bFGF+DKK-1+SB203580 group was higher than that of bFGF+DKK-1 group and bFGF+SB203580 group.(5)EdU test results: Within 24 hours of culture,the number of cell regeneration in positive control group was higher than that in negative control group.The number of cell regeneration in bFGF+DKK-1 group and bFGF+SB203580 group was lower than that in positive control group.The number of cell regeneration in bFGF+DKK-1+SB203580group was higher than that in bFGF+DKK-1 group and bFGF+SB203580 group.(6)Alizarin red staining results: mineralized nodules in positive control group were less than those in negative control group,while bFGF + inhibitors(DKK-1 or SB203580)weremore than those in positive control group,but less than bFGF + inhibitors(DKK-1+SB203580).BCIP/NBT alkaline phosphatase staining results: alkaline phosphatase staining in positive control group was lighter than that in negative control group,while bFGF + inhibitors(DKK-1 or SB203580)were more than that in positive control group.The control group was deep,but shallower than bFGF + inhibitor(DKK-1 +SB203580).(7)The transcriptional levels of osteogenic genes DSPP,ALP,OCN,BSP,OSX and Runx2 were detected by qPCR.The transcriptional levels of bFGF+DKK-1 and bFGF+SB203580groups of DSPP,ALP,OCN,BSP,OSX and Runx2 were up-regulated in varying degrees after 14 days of osteogenesis induction.The transcriptional levels of DSPP,ALP,OCN,BSP,OSX and Runx2 in bFGF+DKK-1+SB203580 groups were down-regulated in varying degrees.In bFGF+DKK-1 group and bFGF+SB203580 group,the gene transcription levels of Nanog,Oct4,Sox2 and Rex1 were down-regulated in varying degrees after 4 days of culture,while those of Nanog,Oct4,Sox2 and Rex1 were down-regulated significantly in bFGF+DKK-1+SB203580 group.Conclusion: bFGF can promote the proliferation of SCAP cells,maintain theirosteogenic differentiation potential and increase the expression of SCAP Stemness genes.Wnt/?-catenin signaling pathway and p38 signaling pathway are involved in the process,and there are interactions.