Nakamura-Ota Miki, Hamanaka Ryoji, Yano Hiroyuki, Adachi Sawako, Sumiyoshi Hideaki, Matsuo Noritaka, Yoshioka Hidekatsu
Department of Matrix Medicine, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Yufu City, Oita, 879-5593, Japan.
Cell Tissue Bank. 2014 Sep;15(3):373-80. doi: 10.1007/s10561-013-9394-9. Epub 2013 Aug 23.
The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.
小鼠前成骨细胞系MC3T3-E1被广泛用于体外研究骨形成和分化。然而，该细胞系在培养过程中不稳定。当前研究旨在建立一个稳定的成骨细胞系。将携带SV 40大T抗原的哺乳动物表达载体导入从新生小鼠颅骨分离的原代培养细胞中。在分离出的细胞系中，选择MN16细胞系进行进一步鉴定。将MN16细胞系培养28天，并与诱导或未诱导的MC3T3-E1细胞系进行比较。使用实时RT-PCR技术检测骨相关基因的表达。茜素红和冯·科萨染色用于检测培养物中结节的矿化。该细胞系在诱导后各种分子标记的基因表达模式以及细胞层中的钙沉积方面表现出成骨细胞的特征。此外，MN16细胞对胶原蛋白的碱性结构域表现出强烈的粘附，这是骨源性细胞特有的结果。发现MN16细胞系在体外能稳定分化为骨形成细胞，应有助于研究骨生物学。
