Thivolet C, Chatelain P, Haftek M, Durand A, Pugeat M
C R Acad Sci III. 1986;303(10):381-6.
Monolayer cell cultures were obtained from a human insulinoma (HIN) after collagenase digestion. HIN cells were initially plated on extracellular matrix (ECM) secreted by bovine corneal endothelial cells. Capsular integrity from cell clusters quickly interrupted and cell began to migrate as adhesive sheets onto ECM. After 2 months on ECM cell attachment and proliferation occurred on plastic allowing cloning of cells by limiting dilution. 9 clones were successfully cultured for 7 months with 20 subsequent passages. Immunoreactivity for insulin by indirect immunofluorescence typical secretory granules by electron microscopy and stable amounts of immunoreactive insulin in culture media suggest that HIN cells are beta cell related. One clone HIN D8 when challenged for half an hour with either 30 mM glucose, 1 mM isobutyl Methylxanthine 4 mM Tolbutamide, 10(-6) M glucagon responded respectively with a 1.5, 2, 3 and 1.5 fold increase in insulin output. Population doubling time of HIN D8 was 42 hrs. Establishment of such insulin secreting cell lines provides a valuable tool for diabetes research.
通过胶原酶消化从人胰岛素瘤（HIN）获得单层细胞培养物。HIN细胞最初接种在牛角膜内皮细胞分泌的细胞外基质（ECM）上。细胞簇的包膜完整性很快被破坏，细胞开始以黏附片的形式迁移到ECM上。在ECM上培养2个月后，细胞在塑料培养皿上附着并增殖，从而可以通过有限稀释法克隆细胞。9个克隆成功培养了7个月，并进行了20次传代。间接免疫荧光法检测胰岛素的免疫反应性、电子显微镜观察典型的分泌颗粒以及培养基中稳定量的免疫反应性胰岛素表明HIN细胞与β细胞相关。一个克隆HIN D8分别用30 mM葡萄糖、1 mM异丁基甲基黄嘌呤、4 mM甲苯磺丁脲、10⁻⁶ M胰高血糖素刺激半小时后，胰岛素分泌量分别增加了1.5倍、2倍、3倍和1.5倍。HIN D8的群体倍增时间为42小时。建立这样的胰岛素分泌细胞系为糖尿病研究提供了一个有价值的工具。
