Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system.
Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system.
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DOI：
10.1083/jcb.112.3.501
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发表时间：
1991-02
期刊：
影响因子：
--
通讯作者：
Landis WJ
中科院分区：
文献类型：
--
作者：
Gerstenfeld LC;Landis WJ
条件被定义为促进细胞生长，肥大，和细胞外基质矿化的培养系统来自鸡胚脊椎软骨细胞。补充抗坏血酸本身导致肥大表型，通过碱性磷酸酶活性和X型合成分别增加10倍和15倍来评估。然而，当培养物在补充有抗坏血酸和20 mM β-甘油磷酸的营养丰富的培养基中生长时，获得了最大的细胞外基质矿化。在3周内的时间研究表明，伴随着几乎恒定的DNA与蛋白质的比例，DNA增加了3-4倍。在此期间，总胶原蛋白从细胞层蛋白的3%增加到20%;总钙和磷含量增加15-20倍。蛋白聚糖的合成最大，直到第12天，但此后表现出四倍的下降。相比之下，总胶原蛋白合成显示大于10倍的增加，直到第18天，结果表明，胶原蛋白合成取代蛋白多糖合成在细胞肥大。单独的分析表明，在整个21天的时间过程中，I型胶原蛋白的合成水平较低。胶原II型和X型的合成增加，在第一个2周的文化，此后，胶原II型的合成下降，而胶原X型的合成继续上升。IX型合成在整个时间过程中保持在不可检测的水平。胶原I型、II型、IX型和X型mRNA和大蛋白聚糖核心蛋白mRNA的水平与其合成水平相关，数据表明合成的翻译前控制。超微结构检查显示细胞和细胞外形态类似于体内发育中的肥大表型。陷窝中的软骨细胞被随机分布的II型胶原纤维（直径约20 nm）和广泛的蛋白多糖形成良好的细胞外基质包围。可以检测到许多泡状结构。培养物可重复矿化，晶体位于细胞外基质中，主要与胶原纤维相关。没有明确的证据表明矿物质与细胞外囊泡相关。该矿物由钙和磷组成的电子探针显微分析，并被确定为一个非常差的结晶羟基磷灰石的电子衍射。总之，这些数据表明，该培养系统由软骨细胞组成，所述软骨细胞通过其升高的碱性磷酸酶和X型胶原水平及其超微结构外观进行体外分化。（400字处截断摘要）
Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta- glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20- fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)