| Objective:(1)Bone marrow mesenchymal stem cells were isolated from the bone marrow of SD rats,cultured and identified.(2)To study the effect of platelet-rich plasma on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats and its mechanism.Methods:(1)Isolation,culture and identification of bone marrow mesenchymal stem cells from SD rats: Bone marrow mesenchymal stem cells were extracted from the long bones of 3-week-old SD rats by whole bone marrow adherent method,and the morphology of the adherent cells was observed and cells were cultured by passage.Flow cytometry was used to detect the expressions of CD29 and CD45 surface antigens of bone marrow mesenchymal stem cells from the 3rd generation SD rats.(2)The effect of platelet-rich plasma on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats: Blood was collected from SD rats,and platelet-rich plasma was extracted by two-step centrifugation,and then activated for experiment.4 groups was divided in this part:(1)control group;(2)5% platelet-rich plasma group;(3)10%platelet-rich plasma group;(4)15% platelet-rich plasma group,The corresponding concentration of platelet-rich plasma was added into the medium and bone formation was induced in each group,respectively.Alkaline phosphatase activity was detected on day 6 and 12,and alizarin red staining was performed on the 18 th day.(3)Mechanism of platelet-rich plasma on osteoblastic differentiation of bone marrow mesenchymal stem cells in SD rats: 3 groups was divided in this part:(1)control group;(2)Experimental group;(3)Inhibition group,5% platelet-rich plasma was added into the medium of the experimental group,and 5% platelet-rich plasma and Wnt/β-catenin signaling pathway inhibitor ICG001 were added into the inhibition group,while bone formation induction was performed.Alkaline phosphatase activity was detected on day6 and 12,alizarin red staining was performed on day 18.On the 6th day,cell proteins and nucleic acids were extracted,and the expressions of the characteristic proteins Wnt3 a and β-catenin of the Wnt/β-catenin signaling pathway and the osteogenic proteins RUNX2 and OPG were detected by Western-blot method.The m RNA expressions of Wnt3 a,β-catenin,RUNX2 and OPG were detected by fluorescence quantitative PCR.Results:(1)Isolation,culture and identification of bone marrow mesenchymal stem cells from SD rats: The cells extracted by whole bone marrow adherence method showed typical morphology of bone marrow mesenchymal stem cells from SD rats.The third generation cells showed high expression of CD29 and low expression of CD45 by flow cytometry.(2)Effect of platelet-rich plasma on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats: The ALP activity of bone marrow mesenchymal stem cells in platelet-rich plasma groups at 5%,10% and 15%concentrations on day 6 of osteogenesis induction was significantly higher than that in control group(P < 0.05),but no significant difference was found among the three groups(P > 0.05).The results of ALP activity on day 12 were similar to those on day 6.On the18 th day of osteogenesis induction,alizarin red staining showed enhanced staining intensity of bone marrow mesenchymal stem cells calcified nodules in the three platelet rich plasma groups,but there was no significant difference among the three groups.(3)Mechanism of platelet-rich plasma on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats: The activity of ALP in the experimental group was significantly higher than control group on the 6th day of osteogenesis induction(P <0.05),and the activity of ALP in the inhibitory group with the addition of Wnt/β-catenin signaling pathway inhibitor ICG001 was significantly lower than experimental group(P < 0.05).The results of ALP activity on the 12 th day were consistent with those on the 6th day.On the 18 th day of osteogenesis induction,alizarin red staining showed that the calcified nodule staining intensity of bone marrow mesenchymal stem cells in the experimental group was stronger than that in the control group,while the calcified nodule staining intensity of the inhibitory group was weaker than that in the experimental group.On the 6th day of osteogenic induction,Western-blot analysis showed that the expressions of Wnt/β-catenin signaling pathway characteristic proteins Wnt3 a and β-catenin and osteogenic related proteins RUNX2 and OPG in the experimental group were significantly increased compared with the control group(P <0.05),while those in the inhibitory group were significantly decreased compared with the experimental group(P < 0.05).The results of q PCR on the 6th day of osteogenesis induction showed that the m RNA relative expression levels of Wnt3 a,β-catenin,RUNX2 and OPG in the experimental group were significantly increased compared with the control group(P < 0.05),while the mrna relative expression levels in the inhibitory group were significantly decreased compared with the experimental group(P< 0.05).Conclusion:(1)Bone marrow mesenchymal stem cells were successfully isolated from SD rats by whole bone marrow adherent method.(2)5%,10%,15% plateletrich plasma can promote osteogenic differentiation of bone marrow mesenchymal stem cell in SD rats,however,the three concentrations of platelet-rich plasma showed no significant difference in promoting osteogenic differentiation.(3)The role of plateletrich plasma in promoting osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats may be related to the Wnt/β-catenin signaling pathway. |
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