Kotobuki Noriko, Matsushima Asako, Kato Youichi, Kubo Yoko, Hirose Motohiro, Ohgushi Hajime
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakouji, Amagasaki, Hyogo 661-0974, Japan.
Cell Tissue Res. 2008 May;332(2):279-88. doi: 10.1007/s00441-008-0580-1. Epub 2008 Mar 4.
To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.
为了研究基质矿化的级联反应，通过用碱性磷酸酶（ALP）抗体进行荧光激活细胞分选，从人成骨样（HOS）细胞中分离出高表达和低表达碱性磷酸酶的细胞。这些细胞从单细胞重新克隆后，在成骨条件下培养，高表达ALP的HOS（H-HOS）细胞出现基质矿化，而低表达ALP的HOS（L-HOS）细胞则没有。众所周知，成骨相关基因之间的相互作用，如 runt 相关转录因子 2（RUNX2）、I 型胶原α1链（COL1A1）、组织非特异性 ALP 和骨钙素（OCN），与基质矿化有关。定量实时聚合酶链反应显示，H-HOS 细胞中 ALP 的基因表达高于 L-HOS 细胞，而 H-HOS 细胞中 RUNX2、COL1A1 和 OCN 的基因表达低于 L-HOS 细胞。当通过转染将这些成骨相关基因的小干扰 RNA（siRNAs）导入 H-HOS 细胞时，只有 ALP siRNA 抑制了基质矿化。此外，不仅 ALP 基因的表达，而且 COL1A1 和 RUNX2 基因的表达也受到 ALP 抑制的影响，尽管 OCN 的表达不受 ALP 抑制的影响。我们已经能够证实，ALP 基因是基质矿化触发因素的有力候选者。这些结果表明，克隆的成骨细胞在研究基质矿化的分子机制方面是有用的，其功能可以通过使用各种 siRNAs 进行调节。
