Quarles L D, Yohay D A, Lever L W, Caton R, Wenstrup R J
Department of Medicine, Duke University Medical Center, Durham, North Carolina.
J Bone Miner Res. 1992 Jun;7(6):683-92. doi: 10.1002/jbmr.5650070613.
We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.
我们研究克隆的小鼠颅骨MC3T3-E1细胞，以确定它们是否表现出与骨组织中成骨细胞相似的发育序列，即未分化的成骨细胞前体增殖，随后是有丝分裂后分化的成骨细胞表型的表达。在发育的初始阶段（培养的第1-9天），MC3T3-E1细胞积极复制，DNA合成率高和细胞数量逐渐增加证明了这一点，但它们保持梭形外观，不表达碱性磷酸酶，也不积累矿化的细胞外胶原基质，这与未成熟的成骨细胞一致。到第9天，培养物呈现立方形形态，达到汇合，并进入生长停滞期。复制的下调与成骨细胞功能的表达相关，包括碱性磷酸酶的产生、前胶原加工成胶原以及胶原细胞外基质的逐渐沉积。细胞外基质的矿化大约在培养16天后开始，标志着成骨细胞表型发育的最后阶段。碱性磷酸酶的表达和矿化是时间依赖性的，而不是密度依赖性的。在发育中的成骨细胞中，I型胶原合成和胶原积累是解偶联的。虽然未成熟细胞中胶原合成和信息表达在第3天达到峰值，但细胞外基质积累极少。相反，在培养7天后，随着胶原生物合成减少，基质积累达到最大。因此，细胞外基质的形成是成熟成骨细胞的功能。抗坏血酸和β-甘油磷酸对于通过碱性磷酸酶和细胞外基质矿化评估的成骨细胞表型表达都是必不可少的。抗坏血酸不会刺激MC3T3-E1细胞中I型胶原基因的表达，但它是细胞外基质中胶原沉积绝对必需的。抗坏血酸还能诱导成熟细胞中的碱性磷酸酶活性，但不能诱导未成熟细胞中的活性。β-甘油磷酸与抗坏血酸显示协同作用，以进一步刺激有丝分裂后、分化的成骨样细胞中的胶原积累和碱性磷酸酶活性。成熟培养物的矿化需要β-甘油磷酸的存在。因此，MC3T3-E1细胞表现出与体内骨形成类似的成骨细胞特征的时间依赖性和顺序性表达。与MC3T3-E1分化相关的发育序列应该为研究介导骨细胞增殖和分化之间转换的信号提供一个有用的模型，也为研究成骨细胞成熟的分子机制和类骨细胞外基质的形成提供一个可再生的培养系统。
