Effect Of Simvastatin On Mrna Of Some Components Of The Wnt Signaling Pathway In The Osteogenic Differentiation Process Of Bone Marrow Stromal Cells Of Rats

Objective:To confirm the stimulating effect of simvastatin on osteogenic differentiation of bone marrow stromal sells(BMSC) of SD rats, and to further study the role of Wnt signaling pathway in this process.Methods: BMSC derived from the tibia and femur of 6-week old famale SD rats were cultured in vitro.Four groups were established: control group(G1),SIM group(G2), RNAi group(G3) and RNAi+SIM group(G4). After the second passage,the cells in G3 and G4 were transfected with the plasmid mediated Axin2 gene.48 hours later,all the cells were treated with osteogenic inductive DMEM and G2 and G4 were added 10-7mol/L SIM meantime.Alkaline phosphatase staining was used 7 days later and von Kossa staining was carried 28 days later to assess osteoblast differentiation.Real time RT-PCR was performed for evaluating the expressions of Axin2, b-catenin, OCN, frizzled-2, Lef-1 and Wnt5a mRNA at day 7 and day 14 after SIM treatment.Results:1. ALP staining:The expression of ALP was significantly higher in G2 than that in G1 and there was no significant difference between G3 and G4.2. von Kossa staining: G2 was much more marked than other groups significantly in matrix mineralization and there was no significant mineralization in G3.3. The results of real time RT-PCR at day 7 after SIM treatment:Comparing with G1,the expression of Axin2 mRNA was significantly lower and frizzled-2 mRNA was significantly higher in G2. Comparing with G1, the expression of b-catenin and frizzled-2 mRNA was significantly lower and OCN,Lef-1 and wnt5a mRNA was significantly higher in G3. The expression of b-catenin mRNA was significantly higher in G4 than that in G3 and the expressions of OCN,Lef-1,Wnt5a mRNA were significantly lower in G4 than those in G3.(P<0.05)4. The results of real time RT-PCR at day 14 after SIM treatment: The expressions of Axin2,OCN,frizzled-2,Lef-1 and Wnt5a mRNA were significantly higher in G2 than those in G1.The expressions of frizzled-2, Lef-1 and Wnt5a mRNA in G3 were significantly higher than those in G1.Comparing with G3,the expression of OCN mRNA was significantly higher and frizzled-2 and Lef-1 mRNA were significantly lower in G4.(P<0.05)Conclusion:1. SIM promoted the osteogenic differentiation of BMSC and changed the expression of mRNA of some components of Wnt signaling.2. When RNAi targeting the mRNA of Axin2 was performed, the activity of Wnt signalling up-regulated and the ability of extracellular matrix to mineralize decreased in the late process of the osteogenic differentiation of BMSC.3. The RNAi targeting the mRNA of Axin2 could not significantly influence the effect that SIM promoted the osteogenic differentiation of BMSC and SIM could accommodate the changed mRNA expression of the components of Wnt signaling caused by RNAi targeting the mRNA of Axin2 to normal level.