The Preliminary Study That RDFCs Promote RBMSCs On Proliferation And Differentia-Tion In Vitro

Objective:The study adopted the co-culture method of allowing full play to cell its part,set up rBMSCs and rDFCs co-culture system, simulate environment in vivo,discuss the proliferation and osteogenic differentiation of rBMSCs co-culture with rDFCs, in order to optimize seed cells of periodontal tissue engineering, so periodontal tissue engineering will do well in the future.Methods:1. Separate mandibula, remove odontotheca under the microscope, obtain primary rDFCs, culture primary rDFCs, transfer of culture and sublime, microanatomy origin authenticate.2. Separate the femur and tibia of hind limb, obtain primary rBMSCs with whole bone marrow adherence method, culture primary rBMSCs, transfer of culture and sublime, microanatomy origin authenticate.3. Set up rBMSCs and rDFCs co-culture system,24 hole plate vaccinate rBMSCs, after 24 hours, Transwell chamber vaccinate rDFCs, co-culture 3,5,7,9 days,CCK8 test the cell proliferation activity of each group rBMSCs.4.6 hole plate vaccinate rBMSCs, no FBS culture 24 hours, cell cycle synchronization, Transwell chamber vaccinate rDFCs,set up rBMSCs and rDFCs co-culture system, co-culture 3,5,7,9days,4 degrees precooling 75% ethanol fixed, FCM test the percentage of S phase and G2 phase cell count in total rBMSCs count.5.12 hole plate vaccinate rBMSCs, after 24 hours, Transwell chamber vaccinate rDFCs, set up rBMSCs and rDFCs co-culture system, set 4 holes, co-culture 7 and 14 days, AKP and BCA kit test OD value, after calculate, rBMSCs AKP activity in cells will be obtained.6.6 hole plate vaccinate rBMSCs, after 24 hours, Transwell chamber vaccinate rDFCs, set up rBMSCs and rDFCs co-culture system,co-culture 10 days, RT-PCR test the expression of osteogenic gene BSP,OCN,COL-I and Runx2.Results:1. Immunohistochemistry authenticated results prove that cells root in ectoblastic mesenchyme, combine morphological observation and draw materials, so the cultured cells are rDFCs.2. Adipogenesis and bone-formation induced, FCM test the expression of specific antigens, combine morphological observation and draw materials, so the cultured cells are rBMSCs.3. CCK-8 and FCM test the proliferation activity of rBMSCs, after co-culture 5 and 7 days, the proliferation activity of experimental group is better than control group, the results are provided with statistics difference.4. Test the AKP activity of rBMSCs, after co-culture 7 and 14 days, the each groups AKP activity in cells is enhancing,compare experimental group to with control group, the results are provided with statistics difference.5. RT-PCR test the expression of rBMSCs osteogenic gene, after co-culture 10 days, experimental group osteogenic gene BSP、OCN、COL-I and Runx2 expression up regulation, compare to control group, the results are provided with statistics difference.Conclusion:After rBMSCs and rDFCs co-culture, the proliferation activity of rBMSCs is increasing, AKP activity in cells is enhancing, osteogenic gene BSP,OCN,COL-I and Runx2 expression up regulation; So, BMSCs have a good future in the periodontal tissue engineering.