Construction The Regulable BMP2 Expression Based On Tet-on System And Its Regulatory Effect On ADSCs

Purpose: The advantage of Tet-on system lies in that its regulation is rigorous, controllable and low background expression which makes it a most potential gene regulation system. Based on the advantages of the Tet-on system, we construct a single plasmid Tet-on system of BMP-2 inducible regulated by Dox. Then, transfect it with the mesenchymal stem cells gathered from rat adipose tissue. And study the influence of the express of BMP-2 among the rat with different degree of the Dox as well as the influence among the ADSCs osteogenic differentiation.Methods: By application of the PCR Technology, to amplification the BMP2 gene in rats and insert it into the Single plasmid Tet-on, then the single plasmid p Tet-BMP2 will be rebuilt. The p Tet-BMP2 along with p MD2.G and ps PAX2 will transfect the 293 T. After 48 and 72 hours, we will collect the concentrated viruses to transfect to ADSCs. Then record the influence of different consistency of Dox to the proliferation and apoptosis of the ADSCs. Use Western Blot technology to testing the relationship between the expression of the BMP2 and the consistency of Dox. Use Alizarin red staining to study the relationship between the numbers of Osteogenic differentiation calcium nodules and the consistency of Dox. By adopt the q PCR method to find the marker gene in the early stage of the osteogenic differentiation and relationship of the relative gene betweeb the expression level of m RNA, the dose of Dox and the induction time.Results: Successfully built the recombinant plasmid of p Tet-BMP2 and the Tet-on slow virus vector which regulate the expression of BMP2 in a very strict way. Both p Tet-BMP2 and Tet-on slow virus vector are successfully transfect the adipose stem cells. There are strict dose dependent relationship between the expression of BMP2 protein and the induction of Dox. When the concentration of Dox is in the 1-5ug/ml, the intracellular expression of BMP2 in the ADSCs cells are increased with the increase of Dox concentration, when the induction of Dox at the dose of 5ug/ml, the expression of BMP2 protein was the highest. The alizarin red staining calcium salt reflect that there are obvious difference between the groups that with Dox and without Dox. Apart from these, the represent areas are more obvious if theconsistency of Dox is increase. q PCR reflects that the expression of the early marker gene of osteogenic differentiation and other relative gene of m RNA are increased with the influence of induction of Dox. And the expression of the early marker gene of osteogenic differentiation and other relative gene of m RNA has a dose dependent relationship with the Dox.Conclusion: This study successfully constructed the ADSCs cell model in the Tet-on control system that strictly regulates the expression of BMP2 in the single particle. And there is a strict dose dependent relationship between the ADSCs osteogenic differentiation behavior and BMP2 expression regulated by the single particle in Tet-on control system.