| Part I:The role of Setd2 in osteogenic differentiation of osteoblasts stimulated by strontium.Aims:The aim of this study is to investigate the role of Setd2 in bone regeneration in femur bone defects and osteogenic differentiation of osteoblasts stimulated by strontium.Materials and Methods:MBG and Sr-MBG were prepared and implanted in the femurs bone defect regions in osteoporosis rats.After 8 weeks of surgery,the new bone regenerated in the bone defect regions were tested using Masson staining and immunohistochemical staining of Runx2.The samples of MBG group and Sr-MBG group were tested by RNA-seq and were analyzed to compare the differentiation of osteogenic genes expression,biological function,histone methylases and demethylases expression.Then the samples were tested with immunohistochemical staining of Setd2 and H3K36me3 to find their expression pattern in the two groups.In addition,immunofluorescence of Runx2 and Setd2,H3K36me3 in bone was performed to co-locate Runx2 with Setd2 and H3K36me3.Human osteoblast cells line MG63 cells were induced by osteoinductive medium containing SrCl2 of different concentrations(0-3mM),then the ALP activity,osteogenic genes expression,and Setd2 expression was performed to choose the optimal concentration of SrCl2.The activity of MAPK pathway and the expression of Setd2 and H3K36me3 were tested by Western blot after MG63 cells were stimulated by SrCl2,of which the total protein,nuclear protein and cytoplasm protein was extracted at different time.Then MG63 cells were pretreated with SCH772984,SP600125 or SB203580,the inhibitors of ERK,JNK and P38,and the expression of Setd2 was tested by Western blot.Setd2 knockdown and overexpression were constructed by lentiviral system,qRT-PCR and Western blot were used to identify the infection efficiency.The concentration of calcification nodes and the expression of osteogenic genes were tested by Alizarin Red staining and RT-PCR following MG63 induced by osteoinducive medium containing SrCl2.Results:The Masson staining showed more mature new bone regeneration in osteoporosis rats femur defects of Sr-MBG groups compared to MBG group.Immuno-histochemical staining showed more frequent Runx2-positive cells in Sr-MBG groups,especially in 5 Sr-MBG group.More osteogenic genes were up-regulated in Sr-MBG group according to the analysis of RNA-seq.The analysis of expression of histone methylases and demethylases showed Setd2 was most up-regulated in Sr-MBG group.In addition,immunohistochemical staining showed the ratio of Setd2-positive and H3K36me3-positive cells were larger in Sr-MBG group.The immunofluorescence staining showed Setd2 and H3K36me3 both expressed in Runx2-positive cells,H3K36me3 only expressed in cell nucleus while Setd2 expressed in both nucleus and cytoplasm.The osteogenic genes and Setd2 expressed highest in MG63 cells induced by induced by osteoinducive medium containing SrCl2 of which the concentration was ImM.Following the stimulation of SrCl2,ERK1/2,P38,JNK in total protein,nuclear protein and cytoplasm protein of MG63 were activated at different time.Moreover,the expression of Setd2 and the level of H3K36me3 were increased after SrCl2 stimulation.However,after blocked ERK,P38,JNK pathways,SrCl2 stimulation failed to promote the expression of Setd2 and the level of H3K36me3.The concentration of calcification nodes and the expression of osteogenic genes were significantly lessened when Setd2 was knocked down in MG63 with the stimulation of SrCl2.Conversely,Setd2 overexpression reinforced the formation of calcification nodes and the expression of osteogenic genes of MG63 stimulated by SrCl2.Apart from this,SrCl2 failed to increase the level of p-ERKl/2 when Setd2 was knocked down in MG63.Conclusion:In the present study,we found that strontium stimulation could up-regulated the expression of Setd2 through MAPK pathway,which may promote osteogenic differentiation of osteoblast cells.Meanwhile,Setd2 also increased the level of p-ERK1/2 to form a positive feedback in this process.Part ?:The role of Setd2 in osteogenic differentiation of osteoblasts stimulated by strontium.Aims:This study aims to investigate the role of Setd2 in periodontal regeneration of periodontal fenestration defect and osteogenic differentiation of periodontal ligament stem cells with the stimulation of strontium.Materials and Methods:MBG and Sr-MBG were prepared and implanted in the periodontal fenestration defect regions in osteoporosis rats.Masson staining and immunohistochemical staining of Runx2 were used to test the new bone regeneration in the bone defect regions after 4 weeks of surgery.Meanwhile,immunohistochemical staining of Setd2,H3K36me3 and hnRNPL of the samples were tested to find their expression pattern in the two groups.The osteoinductive medium containing SrCl2 of different concentrations(0-3mM)was used to induce human periodontal ligament stem cells differentiation,then the ALP activity,proliferation activity and the expression of osteogenic genes expression and Setd2 were tested to choose the optimal concentration of SrCl2.With the stimulation of SrCl2 in corresponding concentration,total protein,nuclear protein and cytoplasm protein of human periodontal ligament stem cells were extracted.The activity of AKT pathway and the expression of Setd2 and hnRNPL were identified by Western blot.Then human periodontal ligament stem cells were pretreated with MK2206,the inhibitors of AKT,and the expression of Setd2 was tested by Western blot.Setd2 knockdown and overexpression were constructed by lentiviral system,qRT-PCR and Western blot were used to identify the infection efficiency.The concentration of calcification nodes,ALP activity and the expression of osteogenic genes were tested by Alizarin Red staining,ALP activity kit and RT-PCR following MG63 induced by osteoinducive medium containing SrCl2.Results:The Masson staining showed more mature new bone regeneration in osteoporosis rat periodontal fenestration defects of Sr-MBG groups compared to MBG group.Immunohistochemical staining showed more frequent Runx2-positive cells in Sr-MBG groups.In addition,the ratio of Setd2-positive and H3K36me3-positive cells were also larger in Sr-MBG group.The osteogenic genes and Setd2 expressed highest in MG63 cells induced by induced by osteoinducive medium containing SrCl2 of which the concentration was ImM.Following the stimulation of SrCl2,AKT in total protein,nuclear protein and cytoplasm protein of human periodontal ligament stem cells were activated after SrCl2 stimulated 5 minutes.Moreover,the expression of Setd2 and the level of H3K36me3 was increased after SrCl2 stimulated 1 hour,while the expression of hnRNPL showed an adverse trend after stimulated 2 hours.However,after blocked AKT pathways,SrCl2 stimulation failed to promote the expression of Setd2 and human periodontal ligament stem cells osteogenic differentiation.The concentration of calcification nodes and the expression of osteogenic genes were significantly lessened when Setd2 was knocked down in human periodontal ligament stem cells with the stimulation of SrCl2 while Setd2 overexpression reinforced the formation of calcification nodes and the expression of osteogenic genes.Conversely,the concentration of calcification nodes and the expression of osteogenic genes were significantly promoted when hnRNPL was knocked down in human periodontal ligament stem cells with the stimulation of SrCl2.Apart from this,the expression of Setd2 was increased when knocked down hnRNPL,and higher after SrCk2 stimulation,while the change of the expression of Setd2 had no influence to the expression of hnRNPL with or without SrCl2 stimulation in human periodontal ligament stem cells.Conclusion:In human periodontal ligament stem cells,Strontium stimulation could activate AKT pathway to reduce the expression of hnRNPL,which may result in the increased expression of Setd2,finally promote osteogenic differentiation. |
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