成年小鼠骨细胞的分离鉴定及其成纤维细胞生长因子受体的表达
发布时间：2018-11-19 10:55
【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。
[Abstract]:Objective to establish a stable method for the isolation and identification of adult mouse bone cells and to detect the expression of its iconic gene and fibroblast growth factor receptor (fibroblast growth factor receptor,FGFR). Methods the tibia and femur of 3-month-old C57 mice were separated and the metaphysis was cut off, then the bone marrow was cut off and cut into about 1 mm fragment. The bone cells were digested alternately with type I collagenase and EDTA solution to obtain bone cells. Bone cells were identified by morphological observation, ALP staining, OC staining and E11/gp38 staining, respectively. QRT-PCR was used to detect the expression of symbolic genes in bone cells and compared with bone cell lines IDG-SW3 and MLO-Y4. The expression of FGFRs in bone cells was detected. Results the isolated osteocytes were dendritic or stellate and connected with each other by synapses. ALP staining showed that osteoblasts were positive, osteoblasts were negative, OC staining was positive for osteoblasts and osteocytes, and fibroblasts were negative. E11/gp38 staining was only positive for bone cells. The expression of osteocyte specific gene DMP1,SOST,FGF23, indicated that osteocytes were isolated successfully, and the expression of FGFR1~3 was different from that of IDG-SW3 and MLOY4 cell lines. FGFR1~3 was expressed in all bone cells. Conclusion the primary bone cells of adult mice were successfully isolated and the expression of FGFR1~3 was found in primary bone cells.
【作者单位】： 第三军医大学大坪医院野战外科研究所:创伤实验室 骨代谢修复中心 创伤烧伤与复合伤国家重点实验室;兰州军区兰州总医院急诊科;第三军医大学大坪医院野战外科研究所康复科;
【基金】：国家重点基础研究发展计划(973计划,2014CB942904) 国家自然科学基金国际合作与交流项目(81220108020) 国家自然科学基金重点项目(81030036) 国家自然科学基金面上项目(81471092)~~
【分类号】：R329.2
[Abstract]:Objective to establish a stable method for the isolation and identification of adult mouse bone cells and to detect the expression of its iconic gene and fibroblast growth factor receptor (fibroblast growth factor receptor,FGFR). Methods the tibia and femur of 3-month-old C57 mice were separated and the metaphysis was cut off, then the bone marrow was cut off and cut into about 1 mm fragment. The bone cells were digested alternately with type I collagenase and EDTA solution to obtain bone cells. Bone cells were identified by morphological observation, ALP staining, OC staining and E11/gp38 staining, respectively. QRT-PCR was used to detect the expression of symbolic genes in bone cells and compared with bone cell lines IDG-SW3 and MLO-Y4. The expression of FGFRs in bone cells was detected. Results the isolated osteocytes were dendritic or stellate and connected with each other by synapses. ALP staining showed that osteoblasts were positive, osteoblasts were negative, OC staining was positive for osteoblasts and osteocytes, and fibroblasts were negative. E11/gp38 staining was only positive for bone cells. The expression of osteocyte specific gene DMP1,SOST,FGF23, indicated that osteocytes were isolated successfully, and the expression of FGFR1~3 was different from that of IDG-SW3 and MLOY4 cell lines. FGFR1~3 was expressed in all bone cells. Conclusion the primary bone cells of adult mice were successfully isolated and the expression of FGFR1~3 was found in primary bone cells.
【作者单位】： 第三军医大学大坪医院野战外科研究所:创伤实验室 骨代谢修复中心 创伤烧伤与复合伤国家重点实验室;兰州军区兰州总医院急诊科;第三军医大学大坪医院野战外科研究所康复科;
【基金】：国家重点基础研究发展计划(973计划,2014CB942904) 国家自然科学基金国际合作与交流项目(81220108020) 国家自然科学基金重点项目(81030036) 国家自然科学基金面上项目(81471092)~~
【分类号】：R329.2
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【共引文献】
相关期刊论文 前5条
1 谷国良;KalervoH. V，
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