HYPERTROPHIC CHONDROCYTES UNDERGO FURTHER DIFFERENTIATION IN CULTURE
HYPERTROPHIC CHONDROCYTES UNDERGO FURTHER DIFFERENTIATION IN CULTURE
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DOI：
10.1083/jcb.117.2.427
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发表时间：
1992-04-01
影响因子：
7.8
通讯作者：
CANCEDDA, R
中科院分区：
文献类型：
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作者：
CANCEDDA, FD;GENTILI, C;CANCEDDA, R
从鸡胚胫骨开始培养，已经确定了促进增生性软骨细胞生长和分化的条件。肥大软骨细胞，在悬浮培养中生长（Castagnola P.， G. Moro， F. Descalzi Cancedda和R. Cancedda, 1986）。J.细胞生物学。102:2310-2317)，当它们达到单细胞阶段时，转移到抗坏血酸存在的底物依赖培养条件下。细胞形态发生变化，变长变平，表达碱性磷酸酶，最终矿化。II型和X型胶原合成停止，取而代之的是I型胶原合成。此外，细胞开始大量产生和分泌一种蛋白，其表观分子质量在还原条件下为82 KD，在非还原条件下为63 KD。该蛋白可溶于酸性溶液，不含胶原结构域，并被糖基化。Ch21蛋白是肥大软骨细胞和骨细胞的标志，在整个培养过程中合成。我们将这个额外的分化阶段定义为成骨细胞样阶段。无论在培养基中添加β -甘油磷酸酯，细胞外基质中的钙沉积都会发生。当细胞以低密度涂覆时，以及当它们已经汇合并在培养中保持不传代达50 d时，都获得了类似的结果。当在第1天至第5天之间将视黄酸添加到肥厚软骨细胞培养中，细胞向成骨细胞样阶段的成熟速度大大加快。2 d后已观察到胶原分泌的转变，3 d后63-kD蛋白的产生。15-20 d后观察到矿化。
Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta-glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.