| Objective:We constructed Fibronectin-1 knockout mice to investigate whether Fn-1 regulates bone homeostasis and osteoporosis by affecting the Wnt signaling pathway.Methods:(1)Bone marrow mesenchymal stem cells specific knockout Fn-1 mouse model was established,and the mouse genotypes were identified by PCR,and the experimental mice(Fn-1fl/fl,Prxl-Cre+),the same litter negative mice(Fn-1fl/fl,Prxl-Cre-)were used as the control group.(2)Total femur protein was extracted,and the expression of target protein and downstream related protein was detected by Western blot assay.(3)MicroCT was used to scan the microstructure of femoral cancellous bone and cortical bone of mice,and to determine the total bone mineral density and bone trabecular density of femur.(4)The fernur of mice was stained with HE,and the histopathological changes and the changes of target proteins and downstream related proteins in tissues were observed by immunofluorescence assay.Results:(1)Bone marrow mesenchymal stem cells specific knockout Fn-1 mouse model was successfully constructed.(2)Compared with the control mice,osteogenic proteins Runx2,Alp,Osx,Ocn and integrin protein Itgbl were highly expressed in the experimental mice(n=3,P<0.05).(3)In the experimental group,the Wnt/β-catenin signaling pathway was activated,and the expression of β-catenin protein was increased(n=3,P<0.05).(4)MicroCT results showed that the number of trabecular bone was increased,the spatial structure was disordered,the trabecular bone and cortical bone were thinned,and the degree of anisotropy was decreased in the experimental group(n=3,P<0.05).Conclusion:(1)The knockdown of Fn-1 in BMSCs can increase the expression of osteoblast-related regulators.(2)Decrease of Fn-1 protein expression can activate the Wnt/β-catenin signaling pathway.(3)The deletion of Fn-1 in bone tissue may be related to the increase of non-load-bearing trabecular bone and the thinning of trabecular bone and cortical bone. |
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