| The hsa-let-7c/IGF-1R axis is composed by hsa-let-7c and IGF-1/IGF-1R.Among them,the IGF axis plays a paramount role in bone formation,maintenance of mineralized skeletons as well as the process of tooth formation,Our previous research results revealed that IGF-1,one of the polypeptide growth factors is the main members of the IGF axis,could significantly enhance the odonto/osteogenic differentiation ofstem cells from apical papilla(SCAPs)and periodontal ligament(PDL)fibroblasts.As a member ofthe let-7 family of mi RNAs,hsa-let-7calso actively participates in the osteogenic differentiation of mesenchymal stem cells.Recently studies have demonstrated that the downregulation of let-7 can upregulate the expression of IGF1 R by mediating the IGF1 signaling cascade to promote cell differentiation.To date,the mechanism for the relationshipbetween the let-7/IGF-1R axis on the odonto/osteogenic differentiation of SCAPs remains unclear.Part I.Isolation and identification of stem cells from apical papillaObjective: To isolate and identify the stem cells from apical papilla.Methods:Impacted third molars(n= 24)were collected from young patients(17–20 years old)in Oral Surgery Department of Jiangsu Provincial Stomatological Hospital after the informed consentwas obtained.The apical papillae were gently detached fromthe immature roots,and SCAPs were obtained by enzyme-digestion method as well as purified by limited dilution.The origin of isolated SCAPs was identified by flow cytometry(FCM).Results: Flow cytometry results revealed that the expressions of STRO-1,CD73,CD90,CD105 were positive,but CD34 and CD45 were negative.Conclusions: The isolated polyclonal apical papilla cells derived from mesenchymal stem cells.Part II.Effects of hsa-let-7c/IGF1 R axis on the proliferation and odonto/osteogenic differentiation of stem cells from apical papilla induced by IGF-1.Objective: To investigate the effects of hsa-let-7c and IGF1/IGF1 R on the proliferation and odonto/osteogenic differentiation of SCAPs.Methods:Theover/lowexpression of hsa-let-7c was transfected into SCAPs respectively,the protein level of IGF1 R and the m RNA level of hsa-let-7c were detected.The proliferation of SCAPs transfected by over/lowexpression of hsa-let-7c stimulated by IGF1 was measured by cellcounting kit-8(CCK8)and FCM.Alizarin red staining and quantitative calcium measurement of SCAPs transfected by over/lowexpression of hsa-let-7c with/without IGF1 stimulation cultured in mineralizationed media were investigated.Moreover,the expression of odonto/osteogenic markers(DMP1/DMP1,DSPP/DSP,ALP,OCN/OCN,OSX/OSX,RUNX2/RUNX2)was measured by real-time RT-PCR and western blot.Results:The protein level of IGF1 R was downregulated and the m RNA level of hsa-let-7c was upregulated in the hsa-let-7c overexpression group,however,the results in the hsa-let-7c lowexpression group were antipodal.The hsa-let-7c and IGF1/IGF1 R had no effect on the proliferation of SCAPs.The hsa-let-7c lowexpression with IGF1 treatment significantly enhanced calcified nodules formation of SCAPs,meanwhile upregulatedboth m RNA and protein expression of odonto/osteogenic markers(DMP1/DMP1,DSPP/DSP,ALP,OCN/OCN,OSX/OSX,RUNX2/RUNX2).Conclusions: The effect of the over/lowexpression of hsa-let-7c transfection into SCAPs was successful.The relationship between the expression of hsa-let-7c and IGF-1R are inverse.The hsa-let-7c and IGF1/IGF1 R had no effect on the proliferation of SCAPs,but the lowexpression of hsa-let-7c with IGF1 treatment,which the level of IGF1 R was upregulated,enhanced their odonto/osteogenic differentiation.Furthermore,identified that IGF1 could modulate the odonto/osteogenic differentiation through IGF1 R.Part III.MAPK pathway involvement in the hsa-let-7c/IGF1 R axis mediated odonto/osteogenic differentiation of SCAPsObjective: To investigate the MAPK pathway mechanism involvement in the hsa-let-7c and IGF1/IGF1 R modulated odonto/osteogenic differentiation of SCAPs.Methods:Theexpression of MAPK pathway-related proteins(p-ERK,ERK,p-p38,p38,p-JNK,JNK)in the overexpression of hsa-let-7cand lowexprssion of hsa-let-7c groups were investigated by western blot respectively in cytoplasm after the over/lowexpression of hsa-let-7c transfected into SCAPs 1 h.Results: The expression of phosphor-JNK and phosphor-p38 was upregulated following the lowexpression of hsa-let-7c transfection(P<0.05),but the level of phosphor-ERK was not affected.The expression of phosphor-JNK and phosphor-p38 was downregulated following the overexpression of hsa-let-7c transfection(P<0.05).Conclusions:The lowexpression of hsa-let-7c with IGF1 treatment,which the level of IGF1 R was upregulated,enhanced odonto/osteogenic differentiation of SCAPs via JNK and p38 MAPK pathway. |
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