高纯度破骨样细胞体外培养及功能表达的研究  

Differentiation and functional expression of highly purified osteoclast-like cells in vitro

作　　者：刘文佳[1] 王晓庚[1] 周洪[1] 李昂 

机构地区：[1]西安交通大学医学院附属口腔医院正畸科 [2]医学研究中心,陕西西安710004

出　　处：《华西口腔医学杂志》2008年第6期599-603,共5页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目(30471911)

摘　　要：目的建立大量高纯度破骨样细胞体外培养的方法,并运用分子生物学技术观察体外诱导培养的破骨样细胞标志酶的基因表达。方法按照巨噬细胞集落刺激因子(M-CSF)30ng/mL、核因子κB受体活化因子配基(RANKL)50ng/mL的质量浓度对骨髓单个核细胞诱导培养6d,利用形态学观察、抗酒石酸酸性磷酸酶(TRAP)染色、Giemsa染色、骨吸收陷窝检测以及破骨细胞标志酶基因表达的检测,对生成的破骨样细胞进行鉴定。结果实验获得的破骨样细胞中,TRAP阳性的单核破骨样细胞多见、TRAP阳性的多核破骨样细胞数量相对较少,胞核从2个到十几个不等;光镜下牙本质片上可见各种形态的骨吸收陷窝;应用RT-PCR方法证实破骨样细胞表达膜型基质金属酶(MT1-MMP)、基质金属蛋白酶-9(MMP-9)、TRAP和组织蛋白酶K(CK)4种标志酶。结论以M-CSF和RANKL作为诱导因子,对大鼠骨髓单个核细胞进行诱导培养可以获得具有典型的破骨细胞形态特征,可以表达特征性标志酶基因,且数量大、纯度高。Objective To establish a culture method for a large amount of highly purified osteoclast-like cells in vitro. To investigate the gene expression of some osteoclast marker enzymes. To lay the foundation for the further study of the signal path on the differentiation and formation of osteoclast-like cells. Methods The bone marrow mononuclear cells of rat were treated with 30 ng/mL macrophagecolony-stimulating factor（M-CSF） and 50 ng/mL receptor activator of NF-κB ligand （RANKL） and cultured for 6 days. After culturing, cells were evaluated by morphology observation, tartrate-resistant acid phosphatase （TRAP） staining, Giemsa staining, pit staining, and the gene expression of some osteoclast marker enzymes. Results The TRAP-positive mononuelear cells were more frequently observed than the multinueleated cells and pit staining could be seen on the dentine slice. The transcription expression of TRAP, matrix metalloproteinase-9（MMP-9）, membrane-typel-matrix metalloproteinase（MT1-MMP） and eathepsin K were detected by RT-PCR. Conclusion The cooperation of M-CSF and RANKL could induce a large amount of highly purified osteoclast-like cells formation in rat bone marrow culture. The typical characteristics of osteoclast-like cells were demonstrated and the enriched osteoclast-like cells expressed TRAP, MMP-9, MT1-MMP and cathepsin K.

关 键 词：破骨样细胞 细胞培养 巨噬细胞集落刺激因子 

分 类 号：R78[医药卫生—口腔医学]