Apoptosis of terminal hypertrophic chondrocytes in an in vitro model of endochondral ossification
Apoptosis of terminal hypertrophic chondrocytes in an in vitro model of endochondral ossification
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DOI：
10.1002/path.1462
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发表时间：
2003-11-01
影响因子：
7.3
通讯作者：
Hillarby, MC
中科院分区：
文献类型：
--
作者：
Cheung, JOP;Grant, ME;Hillarby, MC
生长板软骨细胞在体内软骨内骨化过程中，当其分化到终末肥大阶段时发生凋亡已被广泛接受。在这篇报告中，我们利用一个已建立的哺乳动物软骨内骨化的软骨细胞培养模型来研究软骨细胞在体外进入肥大状态后的命运。胎牛骺软骨细胞用去甲基化剂5-氮杂胞苷处理48 h，然后在氮杂胞苷耗尽的条件下培养。在氮杂胞苷处理的细胞中有凋亡的证据，如核浓缩和碎裂所证明的（第27和35天）使用透射电子显微镜，以及使用荧光标记的膜联蛋白V检测凋亡软骨细胞的质膜表面上暴露的磷脂酰丝氨酸（第27天）。点显示无凋亡的形态学特征。原位杂交研究处理的文化显示，表达的凋亡抑制，bcl-2，始终保持在整个培养期间高，而凋亡诱导剂，Bax，直到第23天才表达。这些数据的定量显示，bcl-2和bax的表达水平的比率随着培养时间的推移，特别是从第23天开始，逐渐转变为有利于bax。两者合计，结果表明，氮杂胞苷处理的骺软骨细胞进入终端肥大从第23天起，在文化和凋亡死亡。这项研究证实了这种培养系统作为一个成功的重演整个哺乳动物软骨细胞分化途径，包括细胞凋亡。该培养模型将被证明是有价值的终末分化软骨细胞的凋亡命运的生长板的研究，以期提供一个更好地了解骨骼畸形和其他病理疾病，如骨关节炎的潜在机制。版权所有（C）2003约翰威利父子有限公司。
It is widely accepted that growth plate chondrocytes undergo apoptosis when they reach the terminal hypertrophic stage of their differentiation during the process endochondral ossification in vivo. In this report, an established chondrocyte cell culture model of mammalian endochondral ossification was utilized to investigate the fate of chondrocytes after they had entered hypertrophy in vitro. Fetal bovine epiphyseal chondrocytes were treated with the demethylating agent, 5-azacytidine, for 48 h and then cultured under azacytidine-depleted conditions. There was evidence for apoptosis in azacytidine-treated cells, as demonstrated by nuclear condensation and fragmentation (days 27 and 35) using transmission electron microscopy, and the detection of exposed phosphatidylserine on the plasma membrane surface of apoptotic chondrocytes (day 27) using fluorescence-labelled annexin V. Treated cultures on days 10 and 20 and untreated cultures at all corresponding time-points showed no morphological characteristics of apoptosis. In situ hybridization studies of treated cultures revealed that expression of the apoptotic suppressor, bcl-2, remained consistently high throughout the culture period, whilst the apoptotic inducer, bax, was not expressed until day 23. Quantification of these data showed a gradual shift in the ratio of the expression level of bcl-2 and bax in favour of bax with time in culture, particularly from day 23 onwards. Taken together, the results indicate that azacytidine-treated epiphyseal chondrocytes entered terminal hypertrophy from day 23 onwards in culture and died by apoptosis. This study confirms this culture system as a successful recapitulation of the entire mammalian chondrocyte differentiation pathway, including apoptosis. The culture model will prove valuable for studies of the apoptotic fate of terminally differentiated chondrocytes in the growth plate with a view to providing a better understanding of the underlying mechanisms of skeletal malformations and other pathological disorders such as osteoarthritis. Copyright (C) 2003 John Wiley Sons, Ltd.