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[目的]比较不同的破骨细胞体外诱导培养方法,为研究外源性因素对骨代谢影响提供方法学基础。[方法]采用骨髓基质细胞和骨髓单核细胞三维共育、骨髓基质细胞培养液转移刺激骨髓单核细胞、重组核因子-κB受体激活物配基(RANKL)诱导骨髓单核细胞和RANKL诱导单核巨噬细胞株RAW264.7四种方法体外诱导破骨细胞。活细胞示踪剂CM-DiI标记RAW264.7后,用激光共聚焦显微镜扫描观察破骨细胞形成过程;抗酒石酸酸性磷酸酶(TRAP)染色法染色破骨细胞。[结果]四种方法均成功地诱导培养出由多个细胞互相融合的TRAP阳性多核破骨细胞。加入RANKL的方法诱导出的破骨细胞较大,数量也较多,方法简单。正常骨髓基质细胞诱导形成的破骨细胞形态较小,数量也相对较少,过程复杂,但可以用来研究干预因素作用于骨髓基质细胞后对破骨细胞分化的间接影响。在对照组及诱导培养体系中均可观察到一类不互相融合的TRAP阳性细胞,含1~3个核,TRAP染色多集中在胞核周围,此类细胞可能是尚未分化的破骨细胞前体。而互相融合的破骨细胞TRAP染色见于整个胞浆。[结论]各种破骨细胞体外诱导培养方法各有优势,应根据不同的实验目的选用不同的破骨细胞诱导培养方法。
[Objective] To compare different methods of osteoclast induction culture in vitro and provide a methodological basis for studying the influence of exogenous factors on bone metabolism. [Methods] BMSCs and bone marrow mononuclear cells were co-cultured in three dimensions. The bone marrow mononuclear cells were stimulated by bone marrow stromal cell culture fluid transfer, and the induction of RANKL and bone marrow mononuclear cells were induced by recombinant human nuclear factor-κB receptor activator ligand (RANKL) Monoclonal Macrophage Cell Line RAW264.7 Four Methods Induce Osteoclasts in. RAW264.7 was labeled with live cell tracer CM-DiI, and the formation of osteoclasts was observed by laser confocal microscopy. The osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). [Results] All the four methods successfully induced the TRAP-positive multinucleated osteoclasts cultured by multiple cells. The method of adding RANKL induced greater osteoclast number, the number is more, the method is simple. The osteoclasts induced by normal bone marrow stromal cells are small in shape, relatively small in number and complicated in process, but can be used to study the indirect effects of intervention factors on osteoclast differentiation after they are applied to bone marrow stromal cells. In the control group and the induction culture system can be observed in a group of TRAP-positive cells do not merge with each other, containing 1 to 3 nuclei, TRAP staining more concentrated in the nucleus, these cells may be undifferentiated osteoclast body. However, TRAP staining of osteoclasts that are integrated with each other is found in the entire cytoplasm. [Conclusion] The methods of in vitro induction culture of various osteoclasts have their own advantages. Different osteoclast induction culture methods should be selected according to different experimental purposes.