Cell-matrix interactions in bone formation and regeneration

为探讨1-羟基-25(OH)_2-维生素D_3(α25(OH)_2-VitD3，VD_3)促进人成骨细胞分化的机制，我们在体外培养人成骨细胞系MG-63，并将其与长效维生素C衍生物L-抗坏血酸-2-磷酸(AsC2-P)单独或单独作用。培养液中加入VD3后，细胞生长速度下降。作为成骨细胞分化早期标志的I型胶原合成和碱性磷酸酶(ALP)活性被VD3和AsC2-P刺激。AsC2-P和VD3的共同存在对细胞的胶原合成和ALP活性有协同作用。在培养液中加入胶原合成抑制剂来抑制胶原合成，可减弱VD3和AsC2-P对ALP活性的刺激作用。转染COL1A1 siRNA表达载体的细胞除…外还降低ALP mRNA的表达水平COL1A1有更多的离子。而在I型胶原包被的培养皿上，细胞的碱性磷酸酶活性显著升高，生长速度明显下降。当细胞培养在涂有热变性胶原蛋白的培养皿中时，这些效应看不到。VD3还上调了成骨细胞分化的关键转录因子Runx2和Osterix以及骨/肝/肾型碱性磷酸酶、I型胶原α1链基因COL1A1和骨钙素等分化标志物的基因表达。正常人成骨细胞和人骨髓间充质干细胞(HBMSC)对VD3的反应非常相似。这些结果表明，VD3刺激的I型胶原基因表达和在ASC2-P存在下产生的成熟I型胶原至少部分参与了ASC2-P和VD3对人成骨细胞分化的刺激作用。Osterix基因的表达使MG-63细胞ALP和COL1A1基因表达水平升高，而Runx2基因表达水平降低。这些结果还表明，VD3通过调节成骨细胞相关转录因子和I型胶原的基因表达来控制成骨细胞的生长和分化，这两种信号的共存是VD3充分表达对人成骨细胞分化的活性所必需的。较少

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