The Expression Of HBMP-2 Gene Induced By Dox In APA Microcapsules

Objective:To study the effects of sodium alginate-poly-L-lysine-sodium alginate (APA) microencapsulated immunoisolation technology on the growth characteristics and differentiations of bone marrow-derived mesenchymal stem cells (BMSCs). To use APA microencapsulation technology to bind up the bone marrow-derived mesenchymal stem cells co-infected by the virus of Adeno-X-TRE-hBMP-2 and the Adeno-X Tet-On. High expression of hBMP-2 gene could be detected with DOX induction. Biological activity of hBMP-2 was tested by osteogenesis of BMSCs. This study could provid sufficient bone formation factor to repair bone defection, and show the feasibility that immunologic rejection was tackled by APA microcapsule, when the BMSCs co-infected by the virus were transplanted.Metheod:1.Adeno-X-TRE-hBMP-2 Plasmid was reproduced in TG1, and then was cellected and identified. To use restriction enzyme PacI to make Adeno-X-TRE-hBMP-2 linearity.2.The linear DNA segment of recombinant Adeno-X-TRE-hBMP-2 was implanted into HEK293 cells by Lipofectamine 2000, and then the infectious recombinant adenovirus particles was gained.3.BD Adeno-X TRE-βgal Control Virus Stock and BD Adeno-X Tet-On virus Stock was amplified in HEK293 cells.4.The best MOI was identfied by the virus of Adeno-X TRE-βgal and Adeno-X Tet-On co-infecting BMSCs.5.BMSCs were co-infected by the virus of Adeno-X-TRE-hBMP-2 and the Adeno-X Tet-On with the optimal MOI. The BMSCs co-infected was wraped in APA microcapsule by the use of the pulsed high-voltage electrostatic microcapsule instrument. The complex was named BMSCs-APA microcapsule Complex.6.The survival rate of BMSCs in APA microcapsule was checked by the stains of FDA and EB.7.The expression of hBMP-2 gene in APA microcapsule was induced by DOX at different concentration (1. 0 ug/ml, 10ug/ml, 100 ug/ml, 200 ug/ml, 300 ug/ml)and detected by WESTERN BLOTING. To analysize the quality-effect relationship between DOX and hBMP2, ELISA was employed to dectect the expression of hBMP-2.8.The osteogenesis of hBMP-2 was tested, whether BMSCs changed to osteoblast under the influence of the hBMP-2.Result:1.The Adeno-X-TRE-hBMP-2 plasmid was confirmed.2.The linear recombinant DNA fragments were constituted successfully.3.High titre AdenoX-TRE-hBMP-2 virus were obtained. The virus titre was 3. 2×108pfu/ml.4.BD Adeno-X TRE-βgal Control Virus Stock and BD Adeno-X Tet-On Virus Stock were amplified respectively, both virus titre were 1. 0×108pfu/ml.5.BMSCs was freed, puryfied and amplified. The growth and differentiation characteristics of the BMSCs in APA microcapsule was unchanged. The survival rate of the BMSCs in APA microcapsule was about 70%-80%. the osteogenesis of the BMSCs out of the APA microcapsule was presented.6.The optimal MOI was 60 pfu/cell.7.The expression of hBMP-2 gene in APA microcapsule was detected by WESTERN BLOTING, when the medium containing DOX was used. DOX inducing concentration and expressing amount of hBMP-2 detected by ELISA demonstrated direct correlation. while DOX>300ug/ml, the quantity of hBMP-2 descenting. While, no DOX no expressing of hBMP-2. 8.The osteogenesis of hBMP-2 was confirmed , when BMSCs was changed to osteoblast under the influence of the hBMP-2.Conclusions:BMSCs were cultured in APA microcapsule successfully and the BMSCs the growth characteristics and differentiations of the BMSCs out of APA microcapsule unchanged. The expression of the hBMP-2 gene in APA microcapsule was well controled by Tet-On expression system. DOX inducing concentration and expressing amount of hBMP-2 demonstrated correlation. The hBMP-2 from the BMSCs-APA microcapsule Complex had biological action.