Gerstenfeld L C, Crawford D R, Boedtker H, Doty P
Mol Cell Biol. 1984 Aug;4(8):1483-92. doi: 10.1128/mcb.4.8.1483-1492.1984.
Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.
在取自11日龄胚胎大腿肌肉的鸡胚胎成肌细胞培养物中，研究了I型和III型前胶原基因的表达。通过添加卵转铁蛋白（30微克/毫升）引发分化，利用相差显微镜进行肉眼观察。在第3天检测到成肌细胞融合和肌管形成，到第7天似乎已完成。通过用[3H]脯氨酸标记细胞培养物1小时，并通过对十二烷基硫酸钠-聚丙烯酰胺凝胶荧光图进行扫描光密度测定来确定前胶原链中的放射性，从而监测前胶原的合成。观察到原α-1（I）、原α-2（I）和原α-1（III）胶原合成速率增加了10至20倍，其中最大增幅发生在第3天至第9天之间。通过Northern印迹和斑点印迹杂交分析检测成肌细胞培养物中的胶原mRNA水平。蛋白质合成速率增加10至20倍的同时，原α-1（I）和原α-2（I）mRNA的稳态水平增加了15倍，原α-1（III）的稳态水平增加了10倍。作为成肌细胞分化过程中胶原表达研究的一个关联，检测了肌动蛋白mRNA的表达。尽管在第4天可检测到α-肌动蛋白，但在所检测的时间段内未观察到从λ和β-肌动蛋白到α-肌动蛋白的完全转变。在妊娠第12天和第17天从胚胎腿部提取的RNA分析中也得到了类似结果。成肌细胞分化表现为肌肉特异性mRNA（如肌动蛋白）以及I型和III型前胶原mRNA的积累。
