兔原代破骨细胞与鼠单核细胞诱导破骨细胞的形态学研究

Comparative morphological observation between rabbit primary osteoclasts and rat monocyte-induced osteoclasts

目的通过提取新生兔骨髓破骨细胞及诱导鼠单核细胞破骨分化,比较原代破骨细胞与诱导形成的破骨细胞之间的形态差异,阐明单核细胞-破骨分化融合过程中的阶段变化特点。方法取2~4 d龄新西兰乳兔骨髓细胞,贴壁法富集原代破骨细胞。取6~8周龄SD大鼠骨髓细胞,M-CSF与RANKL诱导其单核细胞破骨细胞分化。使用HE,Actin-red、Lyso-tracker Green、TRAP染色分别对细胞骨架、溶酶体、抗酒石酸酶进行观察。结果兔原代破骨细胞贴壁速度快,呈现多核(10~50个)煎饼状外观,溶酶体荧光染色呈现强阳性,抗酒石酸酶染色阳性;鼠单核细胞诱导形成的巨噬细胞,贴壁过程较慢,表现为胞质逐渐伸展,细胞折光性逐渐降低,胞浆嗜酸性,溶酶体荧光染色强阳性,细胞内肌动蛋白微丝分布散在。诱导后期TRAP阳性的单核破骨细胞,部分细胞发生融合形成胞质紧贴皿底,形态巨大的多核,细胞骨架染色部分可呈现典型的“封闭环”样结构。结论兔原代破骨细胞与鼠单核细胞诱导形成的破骨细胞形态具有一定相似度,但后者诱导过程中细胞形态变化大,形态复杂多变,在诱导早期阶段可借助细胞光镜下的形态特征,溶酶体荧光染色及细胞骨架染色与杂细胞-间充质细胞鉴别。

Objective To compare the morphological differences between primary osteoclasts and induced osteoclasts and to elucidate the characteristics of changing cascade in rat monocyte-osteoclast inducing process.Methods Primary osteoclasts were isolated from bone marrow of 2-4-day old New Zealand rabbit.Bone marrow cells were extracted from 6-8-week old SD rats.Cells were induced to with M-CSF and RANKL.cells were analyzed with HE staining,Actin-Red,Lyso-Tracker Green,and TRAP staining,respectively.Results The rabbit primary osteoclasts adhered quickly and presented as a pancake-like appearance with multiple nuclei(10-50).Lysosomal fluorescence staining and tartrate-resistant staining(TRAP)were positive.The process of the monocyte-induced macrophage adherence was slow,characterized by its cytoplasmic stretching.Under the stimulation of RANKL,rat macrophages transformed into TRAP-positive mononuclear osteoclasts(OCs).Some cells fused.Cytoplasm of OCs closely attached to the dish bottom.The multinucleated cells were huge with a typical close ring structure.Conclusion Rabbit primary osteoclasts and rat monocyte-induced osteoclasts share similar appearance.While inducing process in the latter is complex.Cells represent with a changeable morphological characteristics.With the tool of lysosomal fluorescence staining and cytoskeleton staining,it can be identified from mesenchymal cells.