Immunological and biochemical studies of collagen type transition during in vitro chrondrogenesis of chick limb mesodermal cells.
Immunological and biochemical studies of collagen type transition during in vitro chrondrogenesis of chick limb mesodermal cells.
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DOI：
10.1083/jcb.73.3.736
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发表时间：
1977-06
期刊：
影响因子：
--
通讯作者：
von der Mark H
中科院分区：
文献类型：
--
作者：
von der Mark K;von der Mark H
这项工作描述了一种方法来监测软骨形成的阶段-24鸡肢中胚层细胞在体外通过分析的发病II型胶原合成与羧甲基纤维素层析，免疫荧光和放射免疫分析。该程序允许在成纤维细胞和成肌细胞存在下对软骨细胞进行特异性和定量测定，两者都合成I型胶原。在组织培养塑料皿和琼脂培养基上，研究了高密度细胞制剂中的软骨发生。发现24期肢体间充质细胞最初仅合成I型胶原。随着软骨形成的开始，观察到逐渐过渡到II型胶原合成。在琼脂上形成的细胞聚集体中，培养1天后开始合成II型胶原，并在6-8天达到总胶原合成的80- 90%的水平。此时，聚集体中心的细胞已获得典型的软骨细胞表型，仅用II型胶原抗体染色，而外周细胞已发育成“软骨膜”，并用I型和II型胶原抗体染色。在每35 mm培养皿中接种5 × 10（6）个细胞的塑料培养皿中，软骨结节在4-6天后形成，但II型胶原的合成仅达到总胶原的10- 20%。大部分细胞分化为成纤维细胞和成肌细胞，并合成I型胶原。这些研究表明，细胞特异性类型的胶原蛋白的分析提供了一个有用的方法，详细说明在体外间充质细胞分化的具体事件。
This work describes an approach to monitor chondrogenesis of stage-24 chick limb mesodermal cells in vitro by analyzing the onset of type II collagen synthesis with carboxymethyl-cellulose chromatography, immunofluorescence, and radioimmunoassay. This procedure allowed specific and quantitative determination of chondrocytes in the presence of fibroblasts and myoblasts, both of which synthesize type I collagen. Chondrogenesis was studied in high-density cell preparations on tissue culture plastic dishes and on agar base. It was found that stage-24 limb mesenchymal cells initially synthesized only type I collagen. With the onset of chondrogenesis, a gradual transition to type II collagen synthesis was observed. In cell aggregates formed over agar, type II collagen synthesis started after 1 day in culture and reached levels of 80-90 percent of the total collagen synthesis at 6-8 days. At that time, the cells in the center of the aggregates had acquired the typical chondrocyte phenotype and stained only with type II collagen antibodies, whereas the peripheral cells had developed into a "perichondrium" and stained with type I and type II collagen antibodies. On plastic dishes plated with 5 X 10(6) cells per 35mm dish, cartilage nodules developed after 4-6 days, but the type II collagen synthesis only reached levels of 10-20 percent of the total collagen. The majority of the cells differentiated into fibroblasts and myoblasts and synthesized type I collagen. These studies demonstrate that analysis of cell specific types of collagen provides a useful method for detailing the specific events in the differentiation of mesenchymal cells in vitro.