Szuts V, Möllers U, Bittner K, Schürmann G, Muratoglu S, Deák F, Kiss I, Bruckner P
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged.
Matrix Biol. 1998 Oct;17(6):435-48. doi: 10.1016/s0945-053x(98)90103-2.
During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.
在软骨内骨形成过程中，新出现的软骨模型中的细胞会经历一系列软骨细胞分化阶段，每个阶段都以特定的基质大分子表达谱为特征，直到最后肥大软骨被骨替代。然而，在许多永久性软骨组织中，由于细胞环境的负调控，软骨细胞不会发生晚期分化。在此，为了解答鸡胚胸骨中软骨细胞命运差异的原因，将来自尾部和头部的细胞分别培养在含有先前确定的可促进或阻止肥大发育的补体的无血清琼脂糖凝胶中。使用适用于琼脂糖培养的新方案提取总RNA，并通过Northern杂交和荧光图像分析监测发育阶段特异性mRNA表达的时间变化。对mRNA积累的动力学研究不仅显示了恢复后头部和尾部培养物表达模式的显著差异，还揭示了与体内软骨发育一致的软骨细胞分化的两个检查点。尾部软骨细胞的终末分化在增殖后期（Ib期）受阻，而头部细胞可自发进行肥大发育。头部软骨细胞的分化是可逆的，因为在胰岛素、成纤维细胞生长因子-2和转化生长因子-β共同作用下，它们可以重新呈现早期增殖（Ia期）表型。因此，后一种培养物中的表达模式类似于关节软骨细胞。我们还提供证据表明，尾部和胸骨软骨细胞从增殖后期（Ib期）进展到肥大期（II期）的能力与其表达印度刺猬基因的不同能力相关。
