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背景:众所周知,骨重建是骨组织中重要的生物学反应过程,其中成骨细胞与破骨细胞发挥了关键作用。但目前,关于骨重建中成骨与破骨细胞间信号传递的深层机制还不清楚。目的:利用transwell技术,在体外建立一种成骨与破骨细胞的新型共育体系,为深入研究骨重建中成骨与破骨细胞的相互作用提供成熟的实验模型。方法:采用MC3T3-E1成骨样细胞株与RAW264.7破骨前体细胞株,进行体外成骨与破骨细胞的诱导分化,并利用Transwell共培养板(0.4μm聚酯膜)建立成骨与破骨细胞的共育体系。共培养6d后,通过测定细胞活性和碱性磷酸酶(ALP)活力分析成骨细胞的增殖和分化活性,利用抗酒石酸酸性磷酸酶(TRAP)染色、甲苯胺蓝(TB)染色、TRAP活性测定及扫描电镜技术观察破骨细胞的分化及骨吸收功能。结果与结论:共培养体系中成骨样细胞的无限增殖能力减弱,而分化活性明显增强,同时破骨前体细胞被诱导分化为成熟的破骨细胞,并具有一定的骨吸收功能。因此,该共培养体系可用于骨重建中成骨与破骨细胞间信号通路的深层研究。
Background: It is well-known that bone remodeling is an important biological reaction in bone tissue. Osteoblasts and osteoclasts play a key role. However, at present, the deep mechanism of signal transduction between osteoblasts and osteoclasts in bone remodeling is still unclear. OBJECTIVE: To establish a new co-culture system of osteoblasts and osteoclasts using transwell technique in order to provide a mature experimental model for the further study of the interaction between osteoblasts and osteoclasts during bone remodeling. METHODS: Osteogenic and osteoclast differentiation in vitro was induced by MC3T3-E1 osteoblast-like cells and RAW264.7 osteoclast precursor cells. Transwell co-culture plates (0.4 μm polyester film) were used to establish osteogenesis Co-culture system with osteoclasts. After 6 days of co-culture, the proliferation and differentiation activity of osteoblasts were assayed by measuring cell viability and alkaline phosphatase (ALP) activity. TRAP staining, toluidine blue (TB) staining and TRAP activity assay And scanning electron microscopy observation osteoclast differentiation and bone resorption function. RESULTS AND CONCLUSION: The osteoblast-like cells in the co-culture system had weaker infinitesimal proliferation ability and enhanced differentiation activity. At the same time, the osteoclast precursor cells were induced to differentiate into mature osteoclasts and had certain bone resorption function. Therefore, this co-culture system can be used for the deep study of osteogenic and osteoclast signal pathways in bone remodeling.