Effects Of Total Flavonoids Of Arachniodes On Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells And Molecular Mechanisms

The first part Effects of Total Flavonoids of Arachniodes onosteogenic differentiation and proliferation of Rat bone marrowmesenchymal stem cellsObjective：To establish a system of cultured Rat bone marrow mesenchymalstem cells (BMSCs) to explore the feasibility of osteoblastic differentiation ofBMSCs in an environment containing Arachniodes total flavonoids in vitro.Methods：BMSCs were harvested from3,4-week-old Sprague Dawley rats bythe method of whole bone marrow differential adhesion. Cell morphology wasobserved under inverted phase contrast microscope and flow cytometry assays surfacemarkers CD44, CD45, CD90and CD11b/c-of P3generation of cell to identify bonemarrow mesenchymal stem cells. BMSCs at passage3were divided into differentgroups according different culture condition. To record morphological and functionalchanges of BMSCs in the experiment at3,6and9days respectively, measuringalkaline phosphatase activity (ALP), mineralized nodules staining, detecting dynamicchange of the mRNA of the gene Col1, OPN, OCN, and OSX.Results：The primary cells after the first exchange of fluid to remove suspendedcells, showed round adherent cells;4d-9d, long spindle or shaped cell rapidlyproliferate, showing radial colony cells;12d-14d, the cell closely packed and fusionShowing a typical the vortex-like or fish-like growth. BMSCs of P3weremorphologically similar to the original cells. CD45and CD11b/c-showed stronglypositive and the positive rate was91.96%and91.0%respectively;The CD90andCD44were negative, the positive rate was only0.32%and1.37%. BMSCs were gooduniformity and purity was above90%. After9days after induction,cell proliferationdecreased,the activity increased,cell transparency decreased, black clumps increased,and it showed cobblestone. With the extension of the osteogenic induction time,intracellular akaline phosphatase activity showed a clear upward trend and the gene transcription and the expression of Osteoblast-specific secretions Col1、OPN、OCNand OSX increased significantly.Conclusion：Total flavonoids of Arachniodes may promote BMSCs to theosteoblast-like cell differentiation, in the differentiation process. The second part molecular mechanisms of Total Flavonoids ofArachniodes on osteogenic differentiation of Rat bone marrowmesenchymal stem cellsObjective: To Research Osteogenic differentiation of rat bone marrowmesenchymal cells(BMSCs) and related signaling pathways.Method: For P3BMSCs, the method is the same of the first part and to detecttranscription and expression of Signal factor mRNA in the Wnt pathway and BMPpathway during the differentiation of BMSCsResults: BMSCs cultured in vitro expressed cell surface markers.TFAinhibited the proliferation of BMSCs, However,it improved BMSCs to differentiateinto bone cells, enhanced the activity of ALP and the formation of calcium nodule,toIncrease WNT pathway and BMP pathways, as well as the transcripts of mRNA ofosteopontin, osteocalcin and collagen I.Conclusion: TFA Promote BMSCs to differentiate into bone cells by the BMPsignaling pathway and Wnt pathway.