The Effect Of Ror2/Wnt5a Signaling Pathway On Adipogenic Differentiation Of Rat Alveolar Bone Marrow Mesenchymal Stem Cells

Objectives The alveolar bone marrow mesenchymal stem cells(BMSCs)with Ror2 silencing was established,detecting the effects of Ror2 silencing and Ror2/Wnt5 a on the expression of BMSCs related genes in rats.The regulation mechanism of Ror2 on adipogenic differentiation of BMSCs in vitro was analyzed in order to find a new therapeutic target for the prevention and treatment of osteoporosis.Methods Alveolar bone of SD rats was isolated under aseptic condition.BMSCs were digested with type I collagenase and Dispase enzyme and identified by flow cytometry.The lentivirus empty vector p Greenpuro-NC and the PGreenpuro-sh Ror2 vector containing sh RNA sequence targeting Ror2 m RNA were constructed.The BMSCs was divided into three groups,namely control group,the empty vector group and Ror2 silencing group.After stable transfection of BMSCs,stable Ror2 silencing cell were obtained.The adipogenic differentiation of the three groups was detected by oil red O staining.The RTq PCR was used to detect the m RNA expression of lipogenic differentiation related factors,which were Wnt5 a,FABP4,LPL and PPAR-? in three groups.Results Flow cytometry showed that the positive rate of CD90 and CD73 was 95.61% and the expression rate of CD45 was lower than 2%.The results showed that the primary cultured cells were bone marrow mesenchymal stem cells(BMSCs).The lentivirus vector was successfully constructed and verified by PCR electrophoresis.After transfection of BMSCs with Ror2 silencing lentivirus,oil red O staining showed that the adipogenic differentiation of the cells was positive.The RT-q PCR analysis showed that the BMSCs with Ror2 silencing could effectively silence the expression of Ror2 gene(P < 0.05)and significantly up-regulate the expression of PPAR-?,FABP4 and LPL(P < 0.05),but not significantly down-regulated the expression of Wnt5 a gene(P > 0.05).Conclusions 1)the recombinant vector of Ror2 silencing gene was successfully constructed and the transfected rat alveolar bone BMSCs could silence gene effectively.2)Ror2 silencing could effectively up-regulate the expression of lipogenic factor PPAR-??FABP4 and LPL in BMSCs and promote its lipid differentiation.3)Ror2 gene silencing in BMSCs is expected potential targets and new strategies for the treatment of osteoporosis.