中文摘要：

目的建立稳定的成年小鼠骨细胞分离和鉴定方法,检测其标志性基因和成纤维细胞生长因子受体（fibroblast growth factor receptor,FGFR）表达。方法将3月龄C57小鼠的胫骨和股骨分离后剪除干骺端,冲去骨髓,切割成约1 mm碎片,用Ⅰ型胶原酶和EDTA溶液交替消化以获取骨细胞。分别通过形态观察、ALP染色、OC染色和E11/gp38染色鉴定骨细胞。qRT-PCR检测骨细胞标志性基因表达,并与骨细胞系IDG-SW3和MLO-Y4比较。检测FGFRs在骨细胞的表达。结果分离出的骨细胞呈多树突状或星状,并借突触彼此连接。ALP染色可见成骨细胞染色阳性,骨细胞染色阴性。OC染色成骨细胞和骨细胞均阳性,成纤维细胞阴性,而E11/gp38染色仅见骨细胞染色阳性。实验所得细胞表达骨细胞特异基因DMP1、SOST、FGF23,表明骨细胞成功分离,且此原代骨细胞与IDG-SW3和MLOY4细胞系表达有差异。FGFR1-3在骨细胞中均有表达。结论成功分离成年小鼠原代骨细胞,发现FGFR1-3在原代骨细胞中均有表达。

英文摘要：

Objective To establish a stable method to isolate and identify primary osteocytes from adult mice,and then detect the expressions of specific genes and fibroblast growth factor receptors（ FGFRs） of the primary osteocytes. Methods The femurs and tibiae of 3-month-old mice were dissected. The epiphyses were cut off,and the marrow was flushed out. The long bones were cut into pieces of 1 mm and digested with collagenase I and EDTA solution alternately. The primary osteocytes were identified by morphological observation,alkaline phosphatase（ ALP） staining,osteocalcin（ OC） staining and E11 / gp38 staining. The expressions of specific genes and FGFRs of the primary osteocytes, IDG-SW3 osteocytes and MLO-Y4 osteocytes were analyzed by qRT-PCR. Results The isolated primary osteocytes were star-shaped and had more dendrites connected with each other. The primary osteocytes showed negative ALP staining,but positive OC and E11 / gp38 staining. Furthermore,these isolated osteocytes also expressed specific genes including DMP1,SOST,and FGF23. FGFR1,FGFR2 and FGFR3 were all expressed in the primary osteocytes.Conclusion The primary osteocytes are isolated successfully from adult mice,and FGFR1,FGFR2 and FGFR3 are all expressed in the primary osteocytes.