Effects Of PTH-FC On Proliferation And Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective:The aim of this study is to evaluate the effects of PTH-FC on osteogenic differentiation of rat bone marrow mesenchymal stem cells,and to screen out the optimal concentration and time of bone marrow mesenchymal stem cells（BMSCs）induced by PTH-FC.Methods:BMSCs were isolated and cultured by using whole bone marrow adherent method.BMSCs were identified by flow cytometry to detect the expression of cell surface markers and cell differentiation into osteoblasts and adipocytes.The effect of proliferation and osteogenic differentiation of BMSCs was studied when it was affected by different concentrations of PTH-FC.The normal medium containing PTH-FC10^-7M,PTH-FC10^-8M and PTH-FC10^-99 M respectively acted on BMSCs,screen out the optimal concentration of the effect of PTH-FC on BMSCs by means of MTT,ALP,and alizarin red staining.BMSCs was induced with PTH-FC10^-99 M osteogenic inducer for 6h（intermittent administration）and 48h（continuous administration）,and to observe the best proliferation promoting and osteogenic differentiation promoting of the effect of PTH-FC on BMSCs by means of MTT,ALP,alizarin red staining and qRT-PCR was used to detect the expression of Runx2,OPN and ALP mRNA.Results:1.BMSCs were successfully isolated and cultured by whole bone marrow adherent method.Flow cytometry showed that negative expression of CD34 and CD45 were 1.85%and1.30%,respectively,the positive expression of CD90 and CD44 were 96.69%and 99.82%,respectively,which proves that the cells are derived from bone marrow mesenchyme.After induced by osteogenic and adipogenic culture for 21 days,obvious calcium nodules and lipid droplets were observed by alizarin red and oil red o staining,It was proved cultured cells are stem cells,which have multiple differentiation potential;2.BMSCs were respectively treated with PTH-FC10^-7M,PTH-FC10^-8M and PTH-FC10^-9M in normal culture medium,The MTT result was showed that PTH-FC promoted the proliferation of BMSCs.The effect of PTH-FC on the proliferation of BMSCs was not obvious at 1d and 2d,and the effect of PTH-FC10^-9M promoted the proliferation of BMSCs at 3d.By ALP and alizarin red staining results seen PTH-FC10^-9M promote osteogenic differentiation of BMSCs,Compared with the control group,but it is not obvious（P>0.05）;therefore,the optimal concentration of PTH-FC for BMSCs was PTH-FC10^-9M;3.Compared with other groups,the osteogenic culture medium of PTH-FC10^-99 M acts on BMSCs for 6 hours（intermittent administration）showed a high ability of osteogenic differentiation,that is,the expression of ALP increased significantly,alizarin red staining showed more calcified nodules and q RT-PCR detection of Runx2,OPN,ALP mRNA expression was significantly higher than the control group,with statistical significance（P<0.05）;and cell proliferation（MTT）results showed no significant difference（P>0.05）between the experimental groups.Conclusions:1.Whole bone marrow adherent method is a simple and effective method for the isolation of BMSCs and can obtain higher purity,the isolated cells have multi-directional differentiation potential;2.The intermittent administration group with PTH-FC10^-99 M acting on BMSCs for 6h has the best ability of osteogenic induction,which suggests it is feasible that PTH-FC can be used as a bone induced growth factor combined with BMSCs to repair bone tissue defects.