| Objective:This exprement is to explore the mechanism of Puerarin on building up bones on different levels.To identify the optimum conditions of the SD rat MSCs cultured with the active component of Radix Puerariae.To observe the effects on proliferation,differentiation to osteoblast of MSCs cultured by various concentrations of Puerarin and confirms the best concentrations and duration of action.To investigate the expression of Cbfα1 mRNA in induced system with Puerarin on molecular biology level.Methods:1.MSCs of SD rats were isolated and purified by using different attachment method,then identified according to morphology and induced-differentiation potentiality.So we can establish a stable culture system in viro.2.Adding different concentrations of Puerarin to MSCs and observing the proliferation between different groups.3.ALP positive cell counts by the improvement Gomori's calcium cobalt staining was explored to observe the osteoblastic differentiation potentiality of MSCs induced by different concentration Puerarin.Blank control is the standard medium without Puerarin and positive control is the same one with dexamethasone,sodiumβ-glycerophosphate and vitamine C. Through these we could confirm the most effective concentration of Puerarin which could induce the cells.4.3 groups were divided:Puerarin group(induced by the most effective concentration),blank control,classic group(classic induce bone formation group,positive control).The ALP activity and the amount of osteocalcin in cells were measured to indicate the osteoblast function of different groups.5.Semi-quantitative analysis was used to detect the expression of Cbfα1 mRNA during osteodifferentiation through RT-PCR method,grouping was same as above. Results:1.MSCs can culture and ahere to the plastic surface in one day and reached confluence in 15 days.In the conditioned induced,MSCs were successfully differentiated into osteoblast and adipose.2.The OD value of MTT showed that 1×10-7mol/L and 1×10-8mol/L were significantly higher than blank groups in the 5 different concentration of Puerarin(P＜0.01).The results expressed that it can increase the quantity of the MSCs.3.The counts of the ALP positive cell showed that Puerarin addition had a definit induction to osteodifferentiation compared with the blank and significant differentiation(P＜0.01).4.The ALP activity sequenced as:classic group＞Puerarin group＞blank control(P＜0.01).5.The expression of the Cbfα1 mRNA followed as:classic group＞Puerarin group＞blank control(P＜0.01).Conclusion:By the different attachment,we can isolate the MSCs from the marrow of the rats' bone. The obtained MSCs were purified and had great capacity of proliferation.They can be purified from generation to generation.In different condition of induced,MSCs can differentiate into osteoblast and adipose.Puerarin could accelerate the proliferation and induce the differentiation to osteoblast of MSCs.The mechanism of Pueraialobata(Wild) Ohwi on preventing and curing osteoporosis may be related to accelerating the proliferation as well as inducing the osteodifferentiation by improving the activity of ALP and enhancing the expression of Cbfα1 mRNA.
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