Study Of The Regulatory-effect Of Transcription Factor Osterix In Osteoblasts Differentiation

Object: To explore a method of isolating and culturing mouse bone marrow mesenchymal stem cell(MSC) and the differentiation of MSC in vitro, analyze the expression of osteogensis association transcription factor Osx, and further study the regulatory mechanism of the early bone maker alphal Type I Collagen (COLLal) by Osx.Methods: We separated and obtained MSC from bone marrow of mouse by gradeient centrifugation in percoll. the effect of different cell density, different concentration of fetal bovine serum(FBS) on MSC proliferation was assessed by MTT assay. After amplified the osx gene through RT-PCR and net PCR, we constructed recombinant plasmid pCDNA3. 1- Osx and transfected to MSC, then biochemistry and histochemistry methods were used to detect ALP activity and the expression of Osx. Finaly, we used electrophoretic mobility shift assay(EMSA) to further study the regulatory mechanism of the early bone maker COLLal by Osx.Results: The best culture conditions for MSC in vitro was 5×10~3cells/cm~2 10%FBS. after induced with 8μg/ml BMP2, MSC differentiated into osteoblast, we can detect higher expression of ALP 6days after induced, after transfection of pCDNA3.1-Osx plasmid, G418-resistant cells were selected , and some of the bone maker was detected. The result of EMSA strongly show that Osx can interreact with the promoter of COLLal.Conclusion: It is a simple effective and pratical method to separate and obtain higher purity and activity of MSC from bone marrow by gradeient centrifugation in percoll. the best culture conditions for MSC was established, and MSC was successfully induced to osteoblast in vitro, further study confirm that Osx play an important role in the process of osteoblast differentiation.