Procollagen synthesis and processing in periodontal ligament in vivo and in vitro. A comparative study using slab-gel fluorography.
Procollagen synthesis and processing in periodontal ligament in vivo and in vitro. A comparative study using slab-gel fluorography.
复制标题
体内和体外牙周膜中原胶原的合成和加工。
DOI：
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发表时间：
1979
期刊：
影响因子：
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通讯作者：
Jaro Sodek
中科院分区：
文献类型：
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作者：
Hardy Limeback;Jaro Sodek
十二烷基硫酸盐/聚丙烯酰胺凝胶电泳和荧光照相相结合已用于定量牙周膜原位和体外牙周膜成纤维细胞合成 I 型和 III 型胶原蛋白。通过引入间断还原步骤实现了 14C 标记的胶原蛋白 α 链的分离，并且 α 链带中的总放射性通过一系列标准曲线与荧光响应相关。从这些曲线可以准确评估合成的 I 型和 III 型胶原蛋白的相对量。同一系统还可以分析各种前胶原的合成和加工。在体内研究中，200 克雄性大鼠被注射 2 mCi [14C]甘氨酸，并在 0.5-6 小时后处死。从下颌磨牙切下牙周韧带，并用 0.45 M 氯化钠提取新合成的胶原蛋白。在体外研究中，在[14C]脯氨酸和[14C]甘氨酸存在下培养汇合的猴牙周膜成纤维细胞。标记胶原蛋白的分析表明，I 型原胶原蛋白快速转化为胶原蛋白，但 III 型胶原蛋白在体外和体内均作为原胶原中间体被回收。胃蛋白酶消化后重复样品的分析表明，III 型胶原蛋白合成占体内合成胶原蛋白总量的 15%，在体外早期传代培养物中占 20%。然而，在传代培养中，成纤维细胞合成的III型比例下降。数据表明，体外成纤维细胞保留了体内发现的胶原合成和前胶原加工的基本特征，但细胞的整体表型表达在培养中并不稳定。
A combination of dodecylsulphate/polyacrylamide gel electrophoresis and fluorography has been used to quantify the synthesis of type I and type III collagens by periodontal ligament in situ and periodontal-ligament fibroblasts in vitro. The separation of 14C-labelled collagen alpha chains was achieved by introducing an interrupted reduction step, and the total radioactivity in the alpha-chain bands related to the fluorographic response by a series of standard curves. From these curves an accurate assessment of the relative amounts of type I and III collagen synthesized could be made. The same system also allowed the synthesis and processing of the respective procollagens to be analyzed. For the study in vivo, 200-g male rats were injected with 2 mCi [14C]glycine and killed 0.5-6 h later. Periodontal ligament was dissected from the mandibular molars and the newly-synthesized collagens extracted with 0.45 M sodium chloride. In the study in vitro, confluent monkey periodontal-ligament fibroblasts were cultured in the presence of [14C]proline and [14C]glycine. Analysis of labelled collagens showed a rapid conversion of type I procollagen to collagen but type III collagen was recovered as a procollagen intermediate both in vitro and in vivo. Analysis of duplicate samples after pepsin digestion showed type III collagen synthesis to comprise 15% of the total collagen synthesized in vivo and 20% in early subcultures in vitro. However, the proportion of type III synthesized by the fibroblasts decreased on subculturing. The data demonstrate that fibroblasts in vitro retain the basic characteristics of collagen synthesis and procollagen processing found in vivo, but the overall phenotypic expression of the cells is not stable in culture.