Transwell小室内建立小鼠成骨-破骨细胞共培养体系

摘要

目的:Transwell小室内建立体外小鼠成骨-破骨细胞共培养体系,并检测体系对成骨及破骨细胞活性的影响.方法:体外培育小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7,RANKL诱导小鼠单核巨噬细胞RAW264.7分化为成熟破骨细胞后,于Transwell小室内建立成骨-破骨细胞共培养体系.通过CCK-8实验、茜素红染色、TRAP染色检测细胞的成骨、破骨活性.采用PCR、Western-Blot方法检测成骨细胞MC3T3-E1中OPG、ALP、RANKL、TGF-b1的基因表达以及RANKL的蛋白表达,检测破骨细胞RANK、NF-κB的基因表达和蛋白表达.结果:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系;共培养体系影响小鼠成骨细胞与破骨细胞的分化活性,镜下可见成骨细胞分化增多,破骨细胞分化稍减少.共培养体系中成骨细胞基因OPG (0.65 ±0.08)、ALP(0.16±0.01)较单独培养OPG(1.00±0.08)、ALP(1.01±0.16)表达下降,而TGF-b1 (4.42±0.21)、RANKL(4.12±1.04)较单独培养组TGF-b1 (1.00±0.10)、RANKL(1.00±0.09)表达上升;破骨细胞相关RANK (0.63±0.06)、NF-κB (0.64±0.08)基因表达较单独培养组的RANK(1.00±0.08)、NF-κB (1.00±0.09)下降,差异均有统计学意义.同时共培养组的OPG (0.43±0.05)、NF-κB (0.59±0.05)的蛋白表达较单独培养组的OPG(0.84±0.06)、NF-κb(1.13±0.03)减少;共培养组RANKL(0.54±0.03)的蛋白表达则较单独培养组的RANKL (0.31±0.03)增加,差异有统计学意义,均与基因表达变化趋势一致.结论:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系,共培养体系中成骨细胞活性高于破骨细胞活性.%Objective:To establish osteoblast-osteoclast cell co-culture system in a Transwell chamber,and detect cell viability of osteoblasts and osteoclasts in system.Methods:Osteoblast MC3T3-E 1 and mouse monocytes RAW264.7 were cultivated in vitro.RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts,osteoblast-osteoclast cell co-culture system was established in Transwell chamber.Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting,Alizarin Red staining,TRAP staining.The expression of OPG,ALP,RANKL,TGF-b 1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR,Western-Blot methods.Also,the expression of RANK,NF-κB in gene and protein level in osteoclast were measured through the same method respectively.Results:The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber.Co-culture system affected cell division activities of osteoblasts and osteoclasts.Differentiation of osteoblasts were increased,while differentiation of osteoclast division were slight decreased under microscope observation.OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16);TGF-b1 (4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1 (1.00±0.10) and RANKL(1.00±0.09) under single culture.However,gene expression of RANK (0.63±0.06) and NF-κB (0.64±0.08) in co-culture system were decreased than RANK (1.00±0.08) and NF-κB (1.00±0.09),in single culture,and had significant differences.Similarly,protein expression of OPG (0.43±0.05) and NF-κB (0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB (1.13±0.03) of single culture.While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL (0.31±0.03),and had statistically differences,which was in agreement of the trend of gene expression change.Conclusion:Co-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber,and the activity of osteoblasts is higher than osteoclasts in co-culture system.