| Objective: To observe the effect of different concentrations of Icariside ?(ICS ?)on osteogenic differentiation and activity of rat bone marrow mesenchymal stem cells(r BMSCs),select the best concentration level of ICS ? that to promote osteogenic differentiation and activity of r BMSCs,and preliminary study on its molecular mechanisms,thus provides theoretical basis for treatment of osteonecrosis of the femoral head(ONFH)of the femoral head by epimedium combined with tissue engineering.Methods: 1.Select 6 SPF young SD rat and take off the neck to death.Separation and extraction r BMSCs by adherent method,and observe cells through light microscope.Induced primary cell,application development,into cartilage induced liquid,liquid into lipid induced r BMSCs between multi-directional differentiation ability appraisal.Use the osteogenic,chondrogenic,adipogenic differentiation medium to identification the force of multiway differentiation of primary r MBSCs.2.Primary rMBSCs culture,passage to P3 and inoculation cells to 12 or 96 orifice.Set up 3 experimental group: low,moderate and high dose,respectively with 1?g/m L,10?g/m L and 100?g/m L ICS ? MEN medium for culturing,the control group contains the same concentration of DMSO.Induced cells to osteogenic differentiation when cells confluence was 60%~70%.After 2 weeks,observe calcified nodules of each group by alizarin red staining;detect the activity of Alkaline Phosphatase(ALP)on 3rd,6th,9th and 12 th day;detect for alkaline phosphatase detection,determination of cell osteogenetic activity;detect the expression of gene related with bone Osterix,Runx-2 at 0th h,4th h,12 th h,24 th h,48 th h,96 th h.According to the dyeing and test result analysis the best concentration leve lof ICS ? that promote the osteogenic differentiation and activity of r BMSCs,and preliminarily stduy its molecular mechanism.Results: 1.The morphological characteristics of r BMSCs that isolated in this experiment were consistent with the morphological characteristics described in the bone cell map at various stages of culture.On the multiway differentiation,the form of r MBSCs gradual changed to different cells and appear calcium deposition,proteoglycan,oil drop.These prove that the cells has a strong ability of multi-directional differentiation.2.Different concentrations of ICS ? intervention osteogenic differentiation of r BMSCs :(1)the alizarin red staining results: the calcium depositionin in experimental groups were significantly higher than the control group.Comparison among the experimental groups shows: the calcium depositionin in moderate dose group was obviously higher than low and high dose group,and there is no obviously different in low and high dose group.(2)The results of ALP detection show that: in 3rd,6th,9th,12 th day,the experimental groups of ALP activity were higher than the control group,and the difference is statistically significant(P < 0.05).In 3rd,6th,9th,12 th day,the ALP activity of moderate dose group were higher than in low and high dose group,and the difference is statistically significant(P < 0.05).There was no statistically significant difference between low and high dose group(P > 0.05).(3)The detection of Osterix expression: experimental groups were higher than control group at different time points,and the difference is statistically significant(P < 0.05).The moderate dose group were higher than low and high dose group,the difference is statistically significant(P < 0.05).Between low and high dose group,at 12 th,the difference is statistically significant(P = 0.044),and at the rest of the time points,the difference has no statistical significance(P > 0.05).(4)The detection of Runx-2 expression: experimental groups were higher than control group at different time points,and the difference is statistically significant(P < 0.05).The moderate dose group were higher than low and high dose group,the difference is statistically significant(P < 0.05).Between low and high dose group,at 48 th,the difference is statistically significant(P = 0.044),and at the rest of the time points,the difference has no statistical significance(P > 0.05).Conclusion: 1.ICS ? has obviously promoting osteogenic differentiation and activity of r BMSCs,and there was obviously a concentration-dependent relationship between them.2.Through the cell staining and the detection of ALP,we can initially determine that the best concentration level of ICS ? that to promote osteogenic differentiation and activity of r BMSCs is 10?g/m L.3.The results of expression of Osterix,Runx-2 confirm described earlier results,and tip ICS ? plays a promote osteogenesis effect between r BMSCs by enhancing Osterix,Runx-2 expression.4.ICS ? can be used directly for the treatment of ONFH by combining with tissue engineering,and the concentration may hopeful be determined to exact value to play a role that obviously promoting osteogenic differentiation and activity of BMSCs. |
PDF Full Text Request