| ABSTRACT ABSTRACT Objective: To establish osteoblast -osteoclast cell co-culture system in a Transwell chamber, and detect of osteoblasts and osteoclasts in the system. Methods: The experiment is divided into single culture group and the co-culture group. Acquire and cultivate in vitro mouse osteoblast MC3T3-E1 and mouse monocytes RAW264.7, RANKL-induced mouse RAW264.7 monocytes division into mature osteoclasts, in a Transwell chamber to establish osteoblast -osteoclast cell co-culture system. By CCK-8 experimenting, Alizarin Red staining, TRAP staining to detect the cell activity of osteoblasts and osteoclasts. By using the PCR and Western blot method, detect the genetic expression of mouse osteoblastic MC3T3-E1 of OPG, TGF-b1 and the protein expression of osteoclasts RANKL, and also to exam the genetic and protein expression of RANK and NF-κb in osteoclasts. Results: The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell may be established in a Transwell chamber. Within the co-culture system the cell division activities of osteoblasts and osteoclasts are significantly enhanced. Under the microscope one can observe the proliferation of the osteoblasts and slight de-acceleration of the osteoclast division. The OPG(0.65±0.08) and ALP(0.16±0.01) gene expression of the co-culture system are less than single culture OPG(1.00±0.08) and ALP(1.01±0.16), and the increase of gene expression in TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of the osteoblasts in the co-culture system than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) of the single culture. However, the gene expression of RANK(0.63±0.06) and NF-κb(0.64±0.08) are decreased than RANK(1.00±0.08) and NF-κb(1.00±0.09), and all of them have statistical difference. Similarly, the protein expression of OPG(0.43±0.05) and NF-κb(0.59±0.05) of the co-culture system are less than the OPG(0.84±0.06) and NF-κb(1.13±0.03) of the single culture. While the RANKL(0.54±0.03) protein expression of co-culture system is more than single culture RANKL(0.31±0.03), and they are statistically differently, which is in agreement of the trend of gene expression change. Conclusion: The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell may be established in a Transwell chamber, the activity of osteoblasts and osteoclasts both change in the system, which the activity of osteoblasts is higher than osteoclasts. in the co-culture system. |
