Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

Effects of Dishevelled Protein DNA Methylation in Wnt Pathway on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Osteoporosis Patients

目的:探讨蓬松蛋白(DVL)DNA甲基化对骨质疏松(OP)患者骨髓间充质干细胞(BMSCs)成骨分化及Wnt通路的影响。方法:分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶(ALP)染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应(RT-PCR)及免疫印迹检测远端缺失同源盒5(Dlx5)、核心结合蛋白因子2(Runx2)、成骨细胞特异性转录因子(OSX)、Ⅰ型胶原蛋白(CollaⅠ)表达。检测DVL1、Wnt、糖原合成酶激酶3(GSK3)、β连环蛋白(β-catenin)表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组(Control组)、甲基转移酶抑制剂组(5-Aza组)、甲基转移酶抑制剂+si-NC组(5-Aza+si-NC组)、甲基转移酶抑制剂+si-Wnt组(5-Aza+si-Wnt组),依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、CollaⅠ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果:成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、CollaⅠm RNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低(P<0.05)。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多(P<0.05),Dlx5、Runx2、OSX、CollaⅠm RNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低(P<0.05)。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少(P<0.05),Dlx5、Runx2、OSX、CollaⅠm RNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高(P<0.05)。结论:DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。

Objective:To investigate the effects of dishevelled protein(DVL)DNA methylation in Wnt pathway on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in osteoporosis(OP)patients.Methods:BMSCs from OP patients were isolated and cultured,BMSCs were cultured for osteogenic induction for 0,7,14 and 21 days,the morphological changes of BMSCs were observed,alkaline phosphatase(ALP)staining and activity were detected,alizarin red staining and calcium nodule formation were detected,the expression of distal deletion homeobox 5(Dlx5),core binding protein factor 2(Runx2),osteoblast-specific transcription factor(OSX)and type I collagen(CollaI)were detected by fluorescence quantitative polymerase chain reaction(RT-PCR)and immunoblotting.The expression of DVL1,Wnt,glycogen synthase kinase 3(GSK3),β-eatenin(β-catenin)and the level of DVL DNA methylation were detected.The methyltransferase inhibitor 5-Aza was added to the osteogenic induction medium,BMSCs were divided into control group(Control group),methyltransferase inhibitor group(5-Aza group),methyltransferase inhibitor+si-NC group(5-Aza+si-NC group)and methyltransferase inhibitor+si-Wnt group(5-Aza+si-Wnt group),ALP activity,alizarin red staining and calcium nodule formation were measured in turn.The levels of Dlx5,Runx2,OSX and Colla 1 were detected by RT-PCR,the protein expression levels of Dlx5,Runx2,OSX,Colla I,DVL1,Wnt,GSK3 andβ-catenin were detected by immunoblotting,and DVL DNA methylation level was detected.Results:BMSCs had strong ALP activity and mineralized nodule formation ability after osteogenic induction,with the increase of culture time,the ALP activity and mineralized nodule formation ability of BMSCs cells increased,the mRNA levels of Dlx5,Runx2,OSX and Colla I and the expression of Wnt,GSK3,β-catenin and DVL1 increased,and DVL DNA methylation level decreased(P<0.05).Compared with Control group,ALP staining was deepened,activity was enhanced,and calcium nodule formation was increased in 5-Aza group(P<0.05),the mRNA and protein expressions of Dlx5,Runx2,OSX,CollaI,Wnt,GSK3,β-catenin and DVL1 were increased,and DVL DNA methylation level was decreased(P<0.05).Compared with 5-Aza+si-NC group,ALP staining was lighter,ALP activity was decreased,calcium nodule formation was decreased in 5-Aza+si-Wnt group(P<0.05),Dlx5,Runx2,OSX,CollaI mRNA and protein expression,Wnt,GSK3,β-catenin,DVL1 expression were decreased,and DVL DNA methylation level was increased(P<0.05).Conclusion:DVL DNA methylation can inhibit the osteogenic differentiation of BMSCs in OP patients by inhibiting Wnt/β-catenin signaling pathway.