Cell-matrix interactions in bone formation and regeneration

为了探讨1α25 (OH)_2维生素D_3 （VD_3）促进人成骨细胞分化的机制，我们对MG-63人成骨细胞系进行了VD_3和/或长效维生素C衍生物l -抗坏血酸2-磷酸（Asc 2-P）存在或不存在的培养。VD_3的存在降低了细胞的生长速度。VD_3和Asc 2-P的存在刺激了I型胶原合成和碱性磷酸酶（ALP）活性，这是早期成骨细胞分化的标志。Asc 2-P和VD_3的共同存在对细胞的胶原合成和ALP活性有协同作用。在培养基中加入胶原合成抑制剂抑制胶原合成，可减弱VD_3和Asc 2-P对ALP活性的刺激作用。sirna表达载体转染COL1A1后，细胞中ALP mRNA的表达量明显低于COL1A1。另一方面，当细胞在I型胶原包被培养皿中培养时，ALP活性显著升高，生长速度减慢。当细胞在涂有热变性胶原蛋白的培养皿中培养时，没有看到这些效应。VD_3还增加了细胞中对成骨细胞分化至关重要的转录因子Runx2和osterix的mRNA水平，以及骨/肝/肾型ALP、COL1A1 （I型胶原α1链的基因）和骨钙素等分化标志物的mRNA水平。正常人成骨细胞和人骨髓间充质干细胞（hBMSC）对VD_3的反应非常相似。这些结果表明，VD_3刺激了I型胶原的基因表达，而在Asc 2-P存在下产生的成熟I型胶原至少部分介导了Asc 2-P和VD_3对这些人成骨细胞分化的刺激作用。MG-63细胞中osterix的表达使ALP和col1a1 mrna水平升高，而Runx2 mrna水平降低。这些结果也表明，VD_3通过调节成骨相关转录因子和I型胶原蛋白的基因表达来控制人成骨细胞的生长和分化，这两种信号的共同存在是VD_3在人成骨细胞分化过程中充分表达活性的必要条件。少

In order to investigate the mechanisms by which 1α25 (OH)_2 vitamin D_3 (VD_3) stimulates the differentiation of human osteoblasts, we cultured MG-63, which is a human osteoblastic cell line, in the presence or absence of VD_3 and/or L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. The cell growth rate was decreased by the presence of VD_3 in the culture medium. Type I collagen synthesis and alkaline phosphatase (ALP) activity, which are markers of early stage osteoblast differentiation, were stimulated by the presence of VD_3 as well as by that of Asc 2-P. The co-presence of Asc 2-P and VD_3 had a synergistic effect on the collagen synthesis and ALP activity of the cells. Inhibition of collagen synthesis by the addition of inhibitors of collagen synthesis to the medium attenuated the stimulative effect of VD_3 and Asc 2-P on the ALP activity. Transfection of the cells with siRNA-expressing vectors for COL1A1 decreased the expression level of ALP mRNA in addit … More ion to that of COL1A1. On the other hand, ALP activity was significantly increased, and the growth rate was decreased, when the cells were cultured on type I collagen-coated dishes. These effects were not seen when the cells were cultured on dishes coated with heat-denatured collagen. VD_3 also increased the mRNA levels for Runx2 and osterix, which are transcription factors critical for osteoblast differentiation, as well as those of differentiation markers such as bone/liver/kidney type ALP,COL1A1, (the gene for the α1 chain of type I collagen), and osteocalcin, in the cells. Normal human osteoblasts and human bone marrow-derived mesenchymal stem cells (hBMSC) showed quite similar responses to VD_3. These results indicate that VD_3 -stimulated gene expression of type I collagen and that mature type I collagen produced in the presence of Asc 2-P mediates at least a part of the stimulative effects of Asc 2-P and VD_3 on the differentiation of these human osteoblastic cells. Levels of mRNAs for ALP and COL1A1were increased, but the level of Runx2 was decreased, by the expression of osterix in MG-63 cells. These results also suggest that VD_3 controls the growth and differentiation of human osteoblastic cells by regulating the gene expression of osteoblast-related transcription factors as well as that of type I collagen, and that the co-presence of both signals is essential for VD_3 to express full activity toward the differentiation of human osteoblasts. Less