Patek C E, Clayton R M
Exp Eye Res. 1985 Mar;40(3):357-78. doi: 10.1016/0014-4835(85)90149-6.
Changes in the differentiation of day-old chick lens epithelium in long-term primary culture conditions were investigated by sodium dodecyl sulphate-polyacrylamide electrophoresis, using integrating densitometry to assess the relative levels of accumulated crystallin and non-crystallin polypeptides and fluorography to assess their relative levels of synthesis. The main changes during the culture period included a relative decline in the proportion of actin and other non-crystallins, an initial increase in 48K delta-crystallin expression followed by a decline and a shift in beta-crystallin expression from a relative preponderance of the 24K and 23K polypeptides to a relative preponderance of the 24K and 22K polypeptides. At all stages the level of the 19K alpha-crystallin was higher than that of the 20K alpha-crystallin polypeptide. In general, the changes in the pattern of expression of these polypeptides in culture were similar to those observed in vivo in the post-hatch chick, suggesting an intrinsic programme of crystallin expression. The changes in gene expression were also tested indirectly by brief exposure of the cells in vitro to a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which is known to produce, in some systems, effects related to the status of the cell at the time of treatment. The effects were found to depend on the stage of differentiation of the culture at the time of treatment. Treatment on day 1 of culture prevented later lentoid formation and severely reduced the expression of all crystallins with the exception of the 34K beta-crystallin polypeptide. Actin was the most abundant soluble cell component, and a proportion of the cells acquired a fibroblast-like morphology. Treatment with MNNG on day 7 led to a delay in lentoid formation and a differential reduction of the synthesis of crystallin polypeptides, whereas the treatment of already differentiated cultures on day 18 and to lesser extents on days 27, 45 and 55, respectively, led to an increase in crystallin synthesis relative to controls. These results suggest that this programme of crystallin gene expression becomes more resistant to change with increasing epithelial differentiation.
采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法，运用积分光密度测定法评估晶状体蛋白和非晶状体蛋白多肽的累积相对水平，并用荧光自显影法评估其合成相对水平，对长期原代培养条件下一日龄雏鸡晶状体上皮细胞的分化变化进行了研究。培养期间的主要变化包括：肌动蛋白和其他非晶状体蛋白的比例相对下降；48Kδ晶状体蛋白表达先增加后下降；β晶状体蛋白表达从相对占优势的24K和23K多肽转变为相对占优势的24K和22K多肽。在所有阶段，19Kα晶状体蛋白的水平均高于20Kα晶状体蛋白多肽的水平。总体而言，这些多肽在培养物中的表达模式变化与雏鸡孵化后体内观察到的变化相似，提示存在晶状体蛋白表达的内在程序。通过体外短暂暴露细胞于致癌物N-甲基-N'-硝基-N-亚硝基胍（MNNG）间接检测基因表达变化，已知在某些系统中，MNNG会产生与处理时细胞状态相关的效应。发现这些效应取决于处理时培养物的分化阶段。培养第1天进行处理可阻止后期晶状体样体形成，并严重降低除34Kβ晶状体蛋白多肽外所有晶状体蛋白的表达。肌动蛋白是最丰富的可溶性细胞成分，部分细胞获得了成纤维细胞样形态。第7天用MNNG处理导致晶状体样体形成延迟，晶状体蛋白多肽合成有差异地减少，而在第18天对已分化培养物进行处理，在第27、45和55天处理程度较轻，分别导致相对于对照的晶状体蛋白合成增加。这些结果表明，随着上皮细胞分化增加，这种晶状体蛋白基因表达程序对变化的抵抗力增强。
