The Experimental Study Of Early Genes Expression In The Process Of Osteogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells

Objective:To isolate and culture bone marrow mesenchymal stem cells(BMSCs) of Sprague Dawley in vitro.BMSCs will be differentiated to osteoblasts by chemical-induced differentiation and the three genes(ALP,BMP-2,Runx2)expression will be monitored in real-time during differentiation.The change patterns of osteoblasts activity of BMSCs will be analysed in the differentiation process.Methods:In vitro,BMSCs are isolated,harvested and amplified from the femur of Sprague Dawley by bone marrow full-adherent.In the third-generation dexamethasone,β-glycerophosphate and vitC are added to induce osteoblasts for one week,next fully culture medium is replaced to harvest it for two weeks.Through phase contrast microscope,drawing the growth curve,flow cytometry,ALP staining,mineralized nodules staining the result will be observed.Using fluorescent probe quantitative real-time polymerase chain reaction(qRT-PCR),in a three-week,three surface genes(ALP,Runx2,BMP-2)expression changes of osteoblasts will be monitored dynamically to analysis the change patterns of osteoblasts activity of BMSCs.Result:BMSCs express CD29.did not express CD45.BMSCs using the improved method of chemical-induced were confirmed as osteoblasts by the morphological observation.ALP staining and mineralized nodules staining.Using qRT-PCR to monitor the results on behalf of the ALP activity of osteoblasts expression that increased after the first 10 days significantly,kept weekly-long high-value.then it declined;at the first 10 days,BMP-2 and Runx2 expression have intersection at a high level.Conclusion:In vitro.BMSCs are isolated,harvested and amplified from the femur of Sprague Dawley by bone marrow full-adherent.The method is simple and effective with good repeatability.With the method of chemical-induced differentiation for one week BMSCs can be differentiate to osteoblasts successfully.The BMSCs induced osteoblasts activity began to enhance from the first 10 days and maintained the high activity in a week;BMP-2 and Runx2 have synergies to promote osteoblast gene expression.