GROWTH-DEPENDENT MODULATION OF TYPE-I COLLAGEN PRODUCTION AND MESSENGER-RNA LEVELS IN CULTURED HUMAN SKIN FIBROBLASTS
GROWTH-DEPENDENT MODULATION OF TYPE-I COLLAGEN PRODUCTION AND MESSENGER-RNA LEVELS IN CULTURED HUMAN SKIN FIBROBLASTS
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DOI：
10.1016/0167-4781(90)90037-3
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发表时间：
1990-06-21
期刊：
影响因子：
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通讯作者：
VUORIO, E
中科院分区：
文献类型：
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作者：
MAKELA, JK;VUORIO, T;VUORIO, E
研究了五种人皮肤成纤维细胞系在不同生长阶段的I型胶原蛋白产生和I型前胶原mRNA水平。将细胞以低密度铺板，并通过快速生长和视觉汇合阶段以每天的间隔跟踪11天，直到培养物达到稳定生长期。每天用[3H]脯氨酸标记一个培养瓶24小时，并分析培养基中放射性标记的I型胶原的产生。计数细胞层，分离细胞质RNA并测定I型前胶原mRNA水平。结果显示，约。2-当细胞达到视觉融合时，前胶原产生和mRNA水平增加一倍。此后，合成速率和mRNA水平保持相对恒定，尽管在进一步培养时观察到这两个参数的下降趋势。结果证实，当细胞培养物用于测量胶原蛋白合成或mRNA水平时，细胞密度的测定是重要的。为了测定前α 2（I）胶原mRNA，使用从人胎儿颅骨提取的RNA构建1193 bp cDNA克隆。克隆的测序揭示了先前公布的序列之间的一些核苷酸和氨基酸差异。这表明前胶原编码序列中存在比预期更多的个体变异。
Five human skin fibroblast lines were studied for type I collagen production and type I procollagen mRNA levels through the different growth phases. The cells were plated at low density and followed for 11 days at daily intervals through the stages of rapid growth and visual confluency until the cultures reached stationary growth phase. Each day one culture flask was labeled with [3H]proline for 24 h, and analyzed for production of radiolabeled type I collagen into culture medium. The cell layers were counted and subjected to isolation of cytoplasmic RNA and determination of type I procollagen mRNA levels. The results revealed an approx. 2-fold increase in procollagen production and mRNA levels when the cells reached visual confluency. Thereafter the synthesis rates and mRNA levels remained relatively constant, although a decreasing tendency of both parameters was observed upon further culturing. The results confirm that determination of cell density is important when cell cultures are used for measurement of collagen synthesis or mRNA levels. For determination of pro.alpha.2(I) collagen mRNA an 1193 bp cDNA clone was constructed using RNA extracted from human fetal calvaria. Sequencing of the clone revealed some nucleotide and amino acid differences between the previously published sequences. This suggests the presence of more individual variation in procollagen coding sequences than expected.