Effect And Mechanism Of Simvastatin On Osteogenic Differentiation Induced By Adipose Mesenchymal Stem Cells In Mice

Objective: 1.Adipose-derived stem cells(ADscs)extracted from mouse groin fat were isolated and cultured,and the osteogenic and adipogenic differentiation potential of ADscs was identified.2.To observe the effects of different simvastatin concentrations on osteogenic differentiation of adipose-derived stem cells(ADSCS)in vitro culture of mice,and to screen the appropriate simvastatin concentration for promoting osteogenic differentiation of ADSCS in vitro experiment.3.To explore the role of Hedgehog signaling pathway in promoting osteogenic differentiation of ADSCs with simvastatin.Research methods: 1.Adipose tissue from the groin and groin region of mice was taken under aseptic conditions to obtain ADSCs for adherent culture.Simvastatin induced osteogenic differentiation and adipogenic differentiation of the third generation(P3)ADSCs.Flow cytometry was used to detect the positive rate of CD29,CD45 and CD90 markers of P3 ADSCs.2.In vitro culture,simvastatin at the concentration of 0,0.01,0.05,0.1,0.5 and 1μmol/L was used to intervene in the osteogenic induction culture of ADSCs of P3 generation mice,and cc K-8 was used to detect the cell proliferation activity for 24 h,72h and 120 h.Alkaline phosphatase activity was detected on the 7th day of culture.The effects of different simvastatin concentrations on cell proliferation activity and alkaline phosphatase were analyzed,and appropriate simvastatin concentration was selected for the next experiment..3.P3 generation ADSC cells with good growth status were selected and randomly divided into four groups.Control group(Ctrl),Simvastatin group(SIM),Cyclopamine group(Cpn)and co-trentment group were selected respectively.ALP detection kit was used to detect the activity of alkaline phosphatase in each group,alizarine red staining was used to detect the osteogenic differentiation of cells,RT-QPCR was used to analyze the expression of osteogenic differentiation related genes in ADSC,and Western blot was used to detect the expression of osteogenic differentiation related genes in ADSC.Results: 1.ADSCs had high expression of mesenchymal stem cell surface markers CD29 and CD90,but low expression of mesenchymal stem cell surface markers CD45;Lipogenic differentiation was induced and orange-red lipid droplets were observed by oil red O staining.After osteogenic differentiation was induced,alizarin red staining showed brick red calcium nodules deposition.2.ADSC of mice were cultured in complete medium containing different concentrations of SIM.Cck-8 cell proliferation detection results showed that there was no significant difference in cell proliferation rate of each group compared with blank control group at 24 h and 72h(P > 0.05).At 120 h,simvastatin culture liquid at 1μmol/L decreased the proliferation rate of the blank control group(P < 0.05),indicating that simvastatin culture liquid at 1μmol/L had a certain inhibitory effect on cell proliferation at 120 h.ADSCs of P3 generation mice were cultured in osteogenic induction medium containing different concentrations of SIM.Results of osteogenic induction showed that the ALP activity of 0.5 umol/L group was significantly higher than that of other groups(P < 0.05),while the ALP activity of 0umol/L group was lower than 1umol/L,but there was no statistical significance(P > 0.05).3.ALP staining and activity detection experiments showed that the ALP activity of ADSC in each group was SIM group,co-treatment group,control group and Cpn group,respectively.The calcium content of ADSC in alizarin red staining and calcium quantitative detection experiment was SIM group,co-treatment group,control group and Cpn group from large to small.PCR and Western blot results showed that the expression of ALP,COL1A1,OCN and RUNX2 genes and related proteins in ADSC of each group were higher in SIM group than in other groups(P < 0.01),higher in co-treatment group than in control group,and lower in Cpn group than in control group.Conclusions: 1.ADSCs extracted from mouse adipose tissue have multidirectional differentiation ability,such as osteogenic differentiation,and high purity,which can be used as seed cells for osteogenic differentiation tissue engineering.2.Low simvastatin concentration had little effect on the proliferation of ADSCs in mice,while high simvastatin concentration inhibited the proliferation of ADSCs.The results of this experiment showed that 0.5μmol/L simvastatin had a better effect on the osteogenic differentiation of ADSCs in mice.3.Simvastatin can partially promote the osteogenic differentiation of mouse ADSCs through the Hedgehog signaling pathway,and this effect can be inhibited by the Hedgehog signaling pathway specific blocker cyclopamine.