| Objective: Bone mesenchymal stem cells(BMSCs)are a kind of adult stem cells with the potential of multilineage differentiation to differentiate into osteoblasts,chondrocytes,adipocytes and so on,which are good speed cells for tissue engineering.The process of osteogenesis and adipogenesis from BMSCs is regulated by various factors.NDRG1 is known as the tumour suppressor,which is regulated by the oncogene N-myc.The effect of NDRG1 on differentiation of bone mesenchymal stem cells is not reproted now.In this study,we aimed to investigate the biological function of NDRG1 on regulating adipgenic and osteogenic differentiation,and preliminarily elucidated the mechanisms.Methods:1.The mRNA experssion level of Ndrg1 was examined in different tissues.We tested the mRNA expression of Ndrg1 in different tissues from the 6-week old C57BL/6J mouse by qRT-PCR,such as heart,slpeen,lung,bone,kidney and so on.2.The effect of NDRG1 on adipogenesis and osteogenesis from bone mesenchymal stem cells was examined.We tested the mRNA expression of Ndrg1 during adipogenic and osteogenic differentiation from the BMSCs and bone marrow stromal cell line ST2.3.We constructed the Ndrg1 plasmid.The Ndrg1 and pcDNA3.1 plasmid were transfected into ST2 cells with adipogenic induction.We observed the adipogenesis from ST2 cells by oil-red O staining.The mRNA expression levels of PPAR?,C/EBP?,aP2 and adipsin were tested by qRT-PCR,the protein expression levels of PPAR??C/EBP? and aP2 were tested by Western blotting.After osteogenic indcucion,ALP staining was used to observe osteogenic differrentiation.The mRNA expression levels of Runx2,Osterix,Alp and Opn were tested by qRT-PCR,the protein expression levels were tested by Western blotting.4.The Ndrg1 siRNA was designed,then transfected into ST2 cells,with adipogenic and osteogenic induction.The differemtiation of ST2 cells were examined by oil-red O staining and ALP staining.The mRNA expression levels of adipocyte and osteoblast specific genes were tested by qRT-PCR,the protein expression levels were tested by Western blotting.5.We constructed the Ndrg1 knockdown lentiviruse.Mouse BMSCs were infected with the Ndrg1 knockdown lentiviruse and pLVX-shRNA2 lentiviruse,with adipogenic and osteogenic induction.The effect of Ndrg1 knockdown lentiviruse on adipogenic and osteogenic differentiation from BMSCs were examined by oil-red O staining,ALP stainin,and qRT-PCR.6.To investigate the relationship between NDRG1 and Wnt/?-catenin singaling,we collected the total protein after transfecting the Ndrg1 plasmid into ST2 cells without induction,then examined the protein expression level of ?-catenin and LRP6 by Western blotting.The translocation of ?-catenin from the cytoplasm into nucleus was detected by cell immunofluorescence assay.Using Western blotting to test the protein experssion of ?-catenin and TCF7L2 in nucleus.7.After transfecting the Ndrg1 siRNA into ST2 cells,then examined the relationship between NDRG1 and Wnt/?-catenin singaling by Western blotting and cell immunofluorescence assay.Results:1.The Ndrg1 mRNA expressed in varoiuse tissues.The mRNA expression of Ndrg1 in mouse intestine was the least,but in kidney and bone was the highest,and in adipose tissue was normal.2.The mRNA expression of Ndrg1 in mouse BMSCs firstly increased then reduced after adipogenic and osteogenic treatment.The mRNA expression of Ndrg1 in ST2 cells firstly increased then reduced after adipogenic treatment,but continuously increased after osteogenic treatment.3.We successfully constructed the Ndrg1 plamid.After transfecting the Ndrg1 plasmid into ST2 cells,with adipogenic induction.NDRG1 promoted ST2 cells differentiate into the adipocytes,the mRNA expression of PPAR?,C/EBP?,aP2 and adipsin were raised,and the protein expression of PPAR?,C/EBP? and aP2 were aslo raised.After treating with osteogenic medium,NDRG1 inhibited ST2 cells differentiate into the osteoblasts,the mRNA and protein expression of Runx2,Osterix,Alp and Opn were reduced.4.The endogenous expression of Ndrg1 was inhibited by Ndrg1 siRNA,NDRG1 inhibited ST2 cells differentiate into the adipocytes.The mRNA expressionof PPAR?,C/EBP?,aP2 and adipsin were reduced,the protein expression of PPAR?,C/EBP? and aP2 were reduced.After osteogenic induction,NDRG1 promoted ST2 cells differentiate into the osteoblasts,the mRNA and protein expression of Runx2,Osterix,Alp and Opn were raised.5.We successfully constructed the Ndrg1 knockdown lentiviruse.Mouse BMSCs were infected with the Ndrg1 knockdown lentiviruse and pLVX-shRNA lentiviruse,with adipogenic induction.The mRNA of PPAR?,C/EBP?,aP2 and adipsin were reduced.However,the Runx2,Osterix and Col1a1 were raised,after ostogenic induction.6.Without induction,the protein expression of ?-catenin and p-LRP6 were reduced after transfecting the Ndrg1 plasmid into ST2 cells.The cell immunofluorescence assay showed that NDRG1 inhibit the translocation of ?-catenin into the nucleus,and the protein expression of ?-catenin and TCF7L2 in nucleus were reduced too.7.The endogenous expression of Ndrg1 was inhibited by Ndrg1 siRNA,the protein expression of ?-catenin and p-LRP6 were raised.The cell immunofluorescence assay showed that the translocation of ?-catenin into the nucleus increased,and the protein expression of ?-catenin and TCF7L2 in nucleus were increased too.Conclusion:1.NDRG1 promoted BMSCs and ST2 cells differrentiate into adipocytes,but inhibited them differentiate into osteoblasts.2.NDRG1 regulated the adipogenic and osteogenic differentiation from BMSCs by the Wnt/?-catenin signaling pathway. |
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