| Objective:To construct a system with osteoporotic rat s bone mesenchymal stem cells(BMSCs)on Wnt3a gene silencing.To research such key signaling molecules as Wnt3a and ?-catenin in Wnt pathway,and to investigate the effects of geraniin on expression of Wnt3a and ?-catenin.Methods:1.Rat's BMSCs were isolated by ovariectomy,and divided in to the three groups:sham group,model group,and 10-9mol/L geraniin-treated group.The effects of geraniin on the osteogenic differentiation of BMSCs were observed by Alizarin red staining,and ALP activity assay,respectively.2.RNA gene chip was used to test BMSCs gene expression profiles,and to screen the expression of up-regulated differential gene.Compared with model group,the key genes related to osteogenic differentiation of BMSCs,changed after treatment of geraniin,were treated by cluster analysis,GO analysis and pathway function distribution.3.The up-regulated key genes in the geraniin-intervented group were determine.For target gene,the three interference sequences with RNAi silencing technology were designed,and the lentivirus were packaged.Using lentivirus as vector to carry the silenced gene to transfect BMSCs,the BMSCs stablely transferred cell lines were esbablished and verified by real time fluorescence quantitative PCR.4.The survival rate of stablely transferred BMSCs was assayed using CCK8 after geraniin treatment for 14 days.The expression of Wnt3a and ?-catenin was tested by use of QPCR and Western blot,respectively.Results1.Alizarin red staining showed that the number of calcium nodules in geraniin(10-9mol/L)group increased significantly,similar to the results of ALP activity test.Geraniin was further verified to promote osteogenic differentiation of BMSCs.2.According to the gene chip data,the KEGG database,and Pathway analysis.Wnt3a was up-regulated target by geraniin intervene.3.Design three interference sequences with RNAi silencing technology or target gene,Sequencing shows the sequence of plasmids constructed was correct.The construction of Wnt3a lentiviral interference plasmid was successful.Transfect the plasmids into BMSCs and Wnt3a gene silencing stable cell line was constructed.4.Compared with group S-Wnt3a,the cell survival rate in group S-Wnt3a+geraniin increased obviously(cP<0.05).;compared with group M-Wnt3a,the cell survival rate in group M-Wnt3a+geraniin also increased obviously.(dP<0.05).5.After Wnt3a silenced,the protein and mRNA expressions of Wnt3a and ?-catenin in BMSCs were both significantly decreased and positively correlated.As compared with group M-Wnt3a,the protein and mRNA expression of Wnt3a and?-catenin increased obviously in group M-Wnt3a+geraniin.(dP<0.05).Conclusions1.Geraniin can obviously promote the osteogenic differentiation of BMSCs,especially the osteogenic differentiation ability of osteoporotic rat BMSCs in vitro.2.For BMSCs,Wnt3a gene silencing inhibites proliferation,andthe expression of Wnt3a and ?-catenin was decreased.3.Geraniin can promote the proliferation of BMSCs which in Wnt3a gene silencing,and increase the expression of Wnt3a and ?-catenin. |
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