| Objective:The previous study of the research group found that the basic fibroblast growth factor can regulate stem cells from the apical papilla osteogenic/odontogenic differentiation and maintain the differentiation potential of Stem cells from the apical papilla(SCAP).The Wnt/β-catenin signaling pathway and the p38 signaling pathway are involved in this process and there is crosstalk.On the basis of previous studies,this experiment further analyzed the molecular mechanism of b FGF in regulating SCAP osteogenic differentiation through transcriptome sequencing,providing theoretical and experimental basis for further research and clinical application of SCAP.Methods: 1.The enzyme digestion method with the original generation of SCAP,by limited dilution method,cloning subculture SCAP,immunofluorescence staining detection ability of multi-directional differentiation of mesenchymal stem cells between appraisal.2.The P3 generation SCAP was divided into two groups: the experimental group(20ng/m L b FGF+osteogenic induction solution),and the control group(osteogenic induction solution),which were induced for 7 days and 14 days respectively.Each group had 3biological replicates and collected samples Total RNA.Transcriptome database of SCAP was constructed by RNA quality inspection,construction of c DNA library and Hi Seq sequencing.Library detection,comparison of genome,gene expression level and differential expression analysis,screening differential expression genes.With differentially expressed genes as the object,gene function annotation,signal pathway enrichment and differentially expressed gene network interaction were further analyzed.3.The threshold value was set as p<0.05 and |log2foldchange|≥1.Transcriptome sequencing results were analyzed and differentially expressed genes were screened for verification by q RT-PCR.According to the above grouping,SCAP was cultured in osteogenic induction fluid containing b FGF and without b FGF for 7 days and 14 days respectively,3 biological replicates were collected for each sample,and the total RNA of each sample was collected,and the relative abundance of differentially expressed genes(MMP13,MMP3,POSTN,FBN2,COL15A1)m RNA was detected with positive-actin as internal reference.Results: 1.Primary SCAP cells were obtained by enzymatic digestion,and colony growth SCAP was observed under the microscope for about 5 days.The cell morphology was short fusiform and the cytoplasm was rich.Immunofluorescence staining results showed that the cell cytoplasm was green after staining with CD24 and wave silk antibody,the cell cytoplasm was red after staining with STRO-1 antibody,and the cytoplasm was not stained with keratin antibody staining;the nuclei were all stained with DAPI;cells Identification of multidirectional differentiation ability: SCAP was stained with alizarin red/oil red O after 28 days of osteogenic and lipid induction,and mineralized nodules/lipid formation could be observed.2.The total RNA from 12 samples was of high purity and good quality.The quality assessment results of RNA-Seq data showed that there were 57,6002,462 read segments in12 samples,with uniform base distribution.By comparing the data with the reference genome,the localization rate of each sample moer than 97%.3.Analysis of differential gene expression showed that 451 genes were significantly expressed in the experimental group and the control group at 7 days(p<0.05 and|log2foldchange|≥1),among which 178 genes were up-regulated and 273 genes down-regulated.A total of 1298 genes were significantly expressed in the experimental group and the control group on day 14,of which 469 were up-regulated and 829 were down-regulated.Venn analysis showed that 86 genes were up-regulated and 144 genes were down-regulated in the 7 and 14 day groups.Through the analysis of genes with significant differentially expressed genes,it was found that the osteogenic differentiation of SCAP by b FGF significantly up-regulated the gene expression levels of MMP13,MMP3 and POSTN,and significantly down-regulated the gene expression levels of FBN2 and COL15A1.4.GO enrichment analysis of differentially expressed genes showed that 50 GO items were significantly enriched by the co-differentially expressed genes of the two groups at 7 and14 days(P< 0.05).The main functions of these differentially expressed genes involved in multicellular biological development,cell conduction,cell differentiation,cell adhesion,extracellular matrix,and active regulation of cell proliferation.KEGG analysis showed that there were significant enrichment pathways of common differentially expressed genes including ECM-receptor interaction,Cytokine-cytokine receptor interaction,Wnt signaling pathway and PI3K-AKT signaling pathway.5.In the 7 and 14 day groups,the common differentially expressed genes were interacted with each other for network analysis,and the key genes were selected according to the size of the nodes: such as PTGS2(COX2),RSPO3,WNT2,BMP3,JAK3 and BCL2A1(up-regulated),FN1,DKK1,RSPO1 and COL15A1(down-regulated),were at the core of the network.These genes are mainly related to bone matrix formation,cell mineralization,Wnt signaling pathway,ECM-receptor interaction,and pi3k-akt signaling pathway.6.q RT-PCR verification experiments showed that compared with the control group,the expressions of MMP13,MMP3 and POSTN were significantly up-regulated(p<0.05),while the expressions of FBN2 and COL15A1 were significantly down-regulated(p<0.05),which was consistent with the results of q RT-PCR detection.These genes were related to collagen degradation,bone mineralization and cell proliferation.Conclusion: The Wnt signaling pathway,the pi3k-akt signaling pathway and the ECM receptor interaction pathway are involved in the b FGF regulation of SCAP osteogenic differentiation.b FGF promotes SCAP cell proliferation,chemotaxis and adhesion,improves SCAP self-renewal ability,while inhibiting apoptosis and osteogenic differentiation ability,maintaining SCAP osteogenic differentiation potential. |
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