Scharfmann R, Corvol M, Czernichow P
Institut National de la Santé et de la Recherche Médicale (Unit 30), Hôpital des Enfants Malades, Paris, France.
Diabetes. 1989 Jun;38(6):686-90. doi: 10.2337/diab.38.6.686.
Pancreatic islets were prepared from 22-day-old rat fetuses. After 5 days of culture in dishes allowing cell attachment, neoformed islets were kept free floating in RPMI-1640 medium (16.5 mM glucose, 1% fetal calf serum). The islets were then pulsed with [3H]leucine and [35S]methionine for 24 h. The conditioned medium was acidified with acetic acid (final pH 2.7), desalted, concentrated, and gel filtered on Bio-Gel P100 in acid conditions. The radioactive material that comigrated with immunoreactive insulinlike growth factor I (IGF-I) produced by the islets was pooled, concentrated, and further characterized by reverse-phase high-performance liquid chromatography on a C18 Bondapak column with a linear gradient of acetonitrile (20-80%). The radioactive material that eluted as pure IGF-I (40% acetonitrile) was further studied by chromatofocusing on a Pharmacia PBE 94 column. A sharp radioactive peak containing [3H]leucine and [35S]methionine was eluted at pH 8.55. This material was immunoprecipitated with an antiserum to IGF-I. This study demonstrated that fetal islet cells synthesize molecules that are, by several criteria, equivalent to native IGF-I.
胰岛取自22日龄的大鼠胎儿。在允许细胞附着的培养皿中培养5天后，新形成的胰岛在RPMI - 1640培养基（16.5 mM葡萄糖，1%胎牛血清）中保持悬浮状态。然后用[³H]亮氨酸和[³⁵S]甲硫氨酸对胰岛进行脉冲标记24小时。将条件培养基用乙酸酸化（最终pH 2.7），脱盐，浓缩，并在酸性条件下于Bio - Gel P100上进行凝胶过滤。将与胰岛产生的免疫反应性胰岛素样生长因子I（IGF - I）共同迁移的放射性物质合并、浓缩，并通过在C18 Bondapak柱上进行反相高效液相色谱进一步表征，流动相为乙腈的线性梯度（20 - 80%）。以纯IGF - I（40%乙腈）形式洗脱的放射性物质通过在Pharmacia PBE 94柱上进行色谱聚焦进一步研究。在pH 8.55处洗脱得到一个含有[³H]亮氨酸和[³⁵S]甲硫氨酸的尖锐放射性峰。该物质用抗IGF - I抗血清进行免疫沉淀。这项研究表明，胎儿胰岛细胞合成的分子在几个标准上等同于天然IGF - I。
