MC3T3-E1Subclone14体外诱导成骨模型的建立及Ano5基因表达的研究

Establishment of an osteoblast model by inducing MC3T3-E1 subclone 14 in vitro and Ano5 gene expression

目的建立MC3T3-E1 Subclone 14体外诱导成骨模型,探讨Ano5基因表达与成骨细胞形成的关系,了解其对成骨分化的影响。方法 MC3T3-E1 Subclone 14细胞贴壁培养,加入条件培养基,分别培养至0d、3d、7d、14d、21d。倒置显微镜下观察细胞形态,通过碱性磷酸酶活力测定和茜素红染色,鉴定成骨细胞。利用REAL TIME-PCR检测Ocn、Colα1、Opg/Rankl、Osterix和Runx2等成骨相关因子的基因表达水平,同时检测Ano5基因在成骨细胞形成过程中的表达趋势。结果体外诱导MC3T3-E1 Subclone 14倒置光学显微镜下观察细胞形态呈梭形。成骨细胞碱性磷酸酶活力逐渐增高。成骨诱导培养至14d和21d,茜素红染色可见细胞表面出现矿化结节。成骨相关因子基因表达均逐渐增高,至21d达到高峰。Ano5基因从诱导分化的第3d开始表达,随后表达量逐渐增高,至14d达到高峰,21d表达量下降。结论在MC3T3-E1 Subclone 14体外诱导分化为成骨细胞过程中,Ano5基因表达量逐渐升高,至14d达到高峰,21d开始表达量具有下降趋势。

Objective To establish an osteoblast model by inducing MC3T3-E1 subclone 14 in vitro, and to examine Ano5 gene expression. Methods Attached MC3T3-E1 subclone 14 cells were cultured with osteoblastic differentiation medium and then harvested at culture day 0, 3, 7, 14 and 21. Cell morphology was observed with reverse microscope. Alkaline phosphatase activity was measured and the cells were stained with Alizarin red S. Expression of osteoblast-related genes such as Ocn, Colα1, Opg/Rankl, Osterix and Runx2 as well as Ano5 gene were examined by real time PCR. Results MC3T3-E1 subclone 14 cells exhibited spindle-shaped after inducing in vitro. Alkaline phosphatase activity was increased gradually. The mineral nodule deposition formed on the surface of cells with Alizarin red S staining at culture day 14 and 21. The expression of osteoblast-related genes was increased significantly with the osteoblastic differentiation culture. The expression of Ano5 gene was enhanced from the third day of differentiation, reached a peak to the 14^th day, and decreased when the cells were cultured to the 21^st day. Conclusion The Ano5 gene expression was increased with the osteoblastic differentiation culture of MC3T3-E1 subclone 14 and reached a peak to the 14^th day and decreased when the cells were cultured to the 21st day.