四种小鼠破骨细胞体外诱导培养方法的比较
Comparison of Four in vitro Osteoclast Culture Systems
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摘要:
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。 Abstract:Objective For studying effects of exogenous factors on bone metabolism,different in vitro osteoclast (OC) culture systems were compared in this study. Methods Bone marrow mononuclear cells co-cultured with bone marrow stromal cells (BMSC) in 3-dimensioal culture system,bone marrow mononuclear cells cultured with bone marrow stromal cells conditional medium,bone marrow mononuclear cells and RAW264.7 cell line cultured in the presence of mouse recombinant receptor activator of nuclear factorkappaB ligand (RANKL) for OC differentiation.RAW264.7 cells were labeled with cross-linkable membrane dye CM-DiI to examine the fu sing process of OC differentiation by Laser Scanning Confocal Microscope.OC was stained by Tartrate Resistant Acid Phosphatase (TRAP). Results All of the procedures successfully developed OC which was indicated by TRAP positive and multinuclear cells fu sing from OC precursors.The OCs induced by exogenous RANKL were easy acquired,whose number and size were more and bigger than those induced by the normal BMSCs.The method of OC differentiation induced by normal BMSCs was complicated,but Was useful for studying the indirect effect of intervention factor on OC differentiation post treated BMSC.Some TRAP positive cells characterized by cell membrane not fusing and TRAP staining only focusing around nucleus were observed in all groups,and contained 1-3 nucleuses.This kind of TRAP positive cells might be OC precursors. Conclusion Each OC differentiation method shows various advantages and which specific procedure is selected should accord to study objectives.
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