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| 诱导小鼠破骨前体细胞成熟分化的实验条件探讨 |
| Study on experimental conditions of mouse osteoclast precursor cell differentiation |
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| DOI： |
| 中文关键词: 破骨细胞 RAW264 7细胞 RANKL |
| 英文关键词:Osteodasts RAW264．7 cells RANKL |
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| 中文摘要: |
| 目的 探讨小鼠单核细胞RAW264.7能否在RANKL诱导下向破骨细胞成熟分化.方法 RANKL作用RAW264.7细胞7天～9天,光镜、透射电镜、扫描电镜(scanning electron microscope,SEM)分别观察其细胞形态学变化,用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法观察TRAP阳性的多核细胞,RT-PCR检测破骨细胞表型和功能基因表达变化情况,扫描电镜观察破骨细胞在骨片上形成骨吸收陷窝.结果 光镜、透射电镜下可见细胞胞体增大,为椭圆形或不规则形,胞核5～10个,扫描电镜下可见细胞表面大量的伪足样突起;此外,RANKL能诱导RAW264.7细胞分化为TRAP染色阳性的多核破骨细胞,细胞多为超过5个核的多核巨细胞;RAW264.7细胞成熟分化后具有骨吸收功能,并且能上调Cathepsin-K、TRAP、RANK等典型破骨细胞表型和功能基因mRNA的表达.结论 RAW264.7细胞是一种较好的破骨前体细胞模型,单用50ng/ml的RANKL体外连续诱导7天以上,能明显促进它向成熟的破骨细胞分化. |
| 英文摘要: |
| Abstract：ObJecave To observe whether RAW264.7 cells.a mouse monocytes cell line，can be induced to differentiate to mature osteoclasts with RANKL．Methods RAW264.7 cells were cultured and treated with RANKL for 7 to 9 days．Cell morphological changes were observed using light microscope，transmissional electron microscope (TEM)，and scanning electron microscope(SEM)．Multinucleated cells were stained with tartrate resistant acid phosphatase(TRAP)and TRAP-positive cells were observed．Expressions of osteoclast phenotype and function genes were detected using Semi-quantitative RT-PCR．Osteodastie resorption pits that formed on the bone slices were observed with SEM．Results Light microscopy and TEM showed that the size of the cells enlarged，and the cell shape Was oval or irregular，and each ceil contained 5 to 10 nucleus．SEM demonstrated that there were large amount of pseudopodia like protrusions on the surface of the cells．Furthermore．RANKL indueed RAW264.7 cells to differentiate to TRAP-positive multinucleated osteoclasts with more than 5 nucleus. The differentiated cells had bone resorption ability．Osteoelast phenotypie and function genes Cathepsin-K，TRAP，and RANK were up—regulated. Conclusion RAW264.7 cells could be used sa a perfect model of osteoclast precursors．RANKL 50ng／mL treatment alone for over 7 days could induce the precursor cells to differentiate to mature osteodasts in vitro． |
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