“脾肾相关”治法SDF-1/CXCR4通路对BMSCs成肌、MDSCs成骨分化的影响

The influence of SDF-1/CXCR4 pathway in BMSCs myogenesis and MDSCs osteogenesis differentiation under different treatment methods based on correlation between spleen and kidney theory

目的探究SDF-1/CXCR4通路在补肾健脾法干预下对骨髓间充质干细胞(BMSCs)成肌、肌源性干细胞(MDSCs)成骨分化的影响。方法正常、诱导(正常血清加相应成骨、成肌诱导液)、补肾阴、补肾阳、健脾、补肾健脾血清干预BMSCs成肌、MDSCs成骨分化培养15 d后取材。免疫荧光鉴定Desmin表达,Image J分析细胞平均荧光强度;茜素红染色观察成骨分化矿化结节;酶联免疫吸附法(ELISA)检测Myosin、BMP2、以及SDF-1、CXCR4蛋白表达;实时荧光定量聚合酶链式反应(Real-time qPCR)检测细胞SDF-1、CXCR4 mRNA表达。结果与正常组相比,BMSCs成肌分化中,Myosin蛋白水平降低(P<0.05)、诱导组Desmin表达升高(P<0.01)、SDF-1、CXCR4蛋白及mRNA表达升高;MDSCs成骨分化中,诱导组出现较小面积的矿化结节,BMP2蛋白表达升高(P<0.05)、SDF-1、CXCR4蛋白及mRNA表达升高。与诱导组相比,BMSCs成肌分化中,用药组Desmin表达升高(P<0.01),Myosin蛋白水平升高(P<0.05)、SDF-1和CXCR4蛋白及mRNA表达水平显著升高;MDSCs成骨分化中,用药组矿化结节数量增多、面积增大,BMP2蛋白水平升高(P<0.05)、SDF-1和CXCR4蛋白及mRNA表达升高。结论补肾健脾中医不同治法可提高SDF-1/CXCR4通路蛋白及mRAN表达从而促进BMSCs成肌、MDSCs成骨分化,补肾健脾法促进作用最为明显。

Objective To investigate the effect of SDF-1/CXCR4 pathway on myogenesis of bone marrow mesenchymal stem cells(BMSCs)and osteogenic differentiation of myogenic stem cells(MDSCs)under the intervention of invigorating kidney and invigorating spleen.Methods Normal serum,conditional serum(normal serum plus corresponding osteogenic and muscle-forming induction fluid),and kidney-Yin,kidney-Yang,invigorating spleen,and invigorating kidney-spleen serum were used in BMSCs muscle formation and MDSCs bone differentiation cultures for 15 days.The expression of Desmin was determined with immunofluorescence.The average fluorescence intensity of cells was analyzed with Image j.Mineralization nodules were observed with Alizarin red staining.The expressions of myosin,BMP2,SDF-1 and CXCR4 were detected with enzyme-linked immunosorbent assay(ELISA).The mRNA expressions of SDF-1 and CXCR4 were detected with real-time quantitative PCR.Results Compared to those in normal group,myosin protein level decreased(P<0.05),Desmin expression increased(P<0.01),and SDF-1,CXCR4 protein and mRNA expressions increased in induction group during myoblast differentiation of BMSCs.In the osteogenic differentiation of MDSCs,small area of mineralized nodules appeared in the induction group.The expressions of BMP2 protein,SDF-1 and CXCR4 protein and mRNA increased(P<0.05).Compared to those in the induction group,Desmin expression,myosin protein level,and SDF-1 and CXCR4 protein and mRNA expression levels increased significantly in the treatment group during myoblast differentiation of BMSCs(P<0.01).During the osteogenic differentiation of MDSCs,the number and area of mineralized nodules increased in the treated group.The protein level of BMP2,protein and mRNA expressions of SDF-1 and CXCR4 increased(P<0.05).Conclusion Kidney-invigorating spleen method enhances the protein and mRNA expressions of SDF-1/CXCR4 pathway,thus promoting BMSCs muscle formation and MDSCs bone differentiation.Kidney-invigorating spleen method has the most obvious promoting effect.