Gillis E, Kluyskens P, Broeckaert D, Coucke P, Nsabumukunzi S, Reyniers P
Acta Otorhinolaryngol Belg. 1980;34(1):23-33.
For the extraction of soft keratins from bovine snout or human epidermis the best results were obtained with a detergent (sodium dodecyl sulphate, SDS) or hydrogen bond breaking agent (urea) in a reducing medium (2-mercaptoethanol, 2-ME). N-acetylcysteine was a little less effective. Keratins from both sources gave typical sets of protein bands with molecular weights between 40.000 and 70.000 daltons. Upon electrofocusing special precautions had to be taken to avoid reoxidation and reaggregation of protein subunits. Keratins from aural cholesteatomas were obtained by extraction with SDA and 2-ME, with N-acetylcysteine alone and to a lesser extent with urea and 2-ME. The pattern of these keratins on SDS-gel is less complicated than that obtained from human skin or bovine snout. Histophotometric results argue for a clear analogy between the nuclear DNA metabolism in normal skin epidermis and cholesteatoma matrix. The only differences of potential relevance are the much wider range of the nuclear DNA amount in cholesteatoma stratum basale cells compared with basal cells in normal epidermis, and the higher persistence of nuclear DNA in cholesteatoma stratum granulosum, indicating postponement of keratinocyte terminal differentiation.
从牛鼻或人表皮中提取软角蛋白时，在还原介质（2-巯基乙醇，2-ME）中使用去污剂（十二烷基硫酸钠，SDS）或氢键断裂剂（尿素）可获得最佳结果。N-乙酰半胱氨酸的效果稍差。来自这两种来源的角蛋白产生了典型的蛋白质条带，分子量在40,000至70,000道尔顿之间。在等电聚焦时，必须采取特殊预防措施以避免蛋白质亚基的再氧化和再聚集。耳胆脂瘤中的角蛋白是通过用SDA和2-ME提取、单独使用N-乙酰半胱氨酸以及较少程度地使用尿素和2-ME获得的。这些角蛋白在SDS凝胶上的图谱比从人皮肤或牛鼻中获得的图谱要简单。组织光度测量结果表明，正常皮肤表皮和胆脂瘤基质中的核DNA代谢之间存在明显的相似性。唯一可能相关的差异是，与正常表皮中的基底细胞相比，胆脂瘤基底层细胞中的核DNA量范围要宽得多，并且胆脂瘤颗粒层中的核DNA持续时间更长，这表明角质形成细胞的终末分化延迟。
