Brubaker P L, Vranic M
Endocrinology. 1987 May;120(5):1976-85. doi: 10.1210/endo-120-5-1976.
To establish an in vitro model to investigate the glucagon-related peptides, fetal rat intestinal cells were enzymatically dispersed and placed into culture for up to 7 days. After 1 day in culture, the presence of epithelial-like cells containing glucagon-like immunoreactivity (GLI) was demonstrated using immunocytochemical techniques. The cell peptides were extracted by passage through a cartridge of octadecylsilyl silica and characterized by gel filtration and RIA. Two GLI moieties were detected with apparent mol wts of 11,000-12,000 and 5,000-6,000. The immunoreactive profile obtained for the cells in culture was identical to that of both whole fetal rat intestine and adult rat ileum. The presence of glucagon could not be demonstrated in any of the extracts. The basal levels of GLI and apparent immunoreactive glucagon (IRGa) were 1,457 +/- 381 and 198 +/- 57 pg/dish, respectively, on day 1 of culture. The GLI content of the cells, but not the IRGa, declined with time in culture for up to 5-7 days (P less than 0.03). Addition of insulin to the culture medium (10 or 100 mU/ml) did not influence the decrease in GLI content of the cells, but did inhibit the production of IRGa (P less than 0.05). Addition of 500 mg/dl glucose to the cells in the presence of 20 microU/ml insulin increased the secretion of GLI by 42 +/- 7% over 2 h (P less than 0.05). The stimulation by glucose was not seen in the absence of insulin or with higher insulin concentrations (100 microU/ml), nor did insulin alone (100 microU/ml) have any effect on the release of GLI. Thus, fetal rat intestinal cells in culture produce the GLI peptides, and secrete them in response to glucose. This system may provide a means by which the synthesis and control of secretion of the glucagon-related peptides can be investigated.
为建立一个体外模型来研究胰高血糖素相关肽，将胎鼠肠道细胞用酶分散并培养长达7天。培养1天后，采用免疫细胞化学技术证实存在含胰高血糖素样免疫反应性（GLI）的上皮样细胞。细胞肽通过十八烷基硅烷硅胶柱进行提取，并通过凝胶过滤和放射免疫分析进行表征。检测到两个GLI部分，其表观分子量分别为11,000 - 12,000和5,000 - 6,000。培养细胞获得的免疫反应谱与整个胎鼠肠道和成年大鼠回肠的免疫反应谱相同。在任何提取物中均未证实有胰高血糖素存在。培养第1天时，GLI和表观免疫反应性胰高血糖素（IRGa）的基础水平分别为1,457±381和198±57 pg/培养皿。培养细胞中GLI的含量随培养时间长达5 - 7天而下降（P＜0.03），但IRGa含量未下降。向培养基中添加胰岛素（10或100 mU/ml）不影响细胞GLI含量的下降，但确实抑制了IRGa的产生（P＜0.05）。在存在20 μU/ml胰岛素的情况下，向细胞中添加500 mg/dl葡萄糖可使GLI在2小时内的分泌增加42±7%（P＜0.05）。在无胰岛素或胰岛素浓度较高（100 μU/ml）时未观察到葡萄糖的刺激作用，单独的胰岛素（100 μU/ml）对GLI的释放也没有任何影响。因此，培养的胎鼠肠道细胞产生GLI肽，并对葡萄糖作出反应而分泌它们。该系统可能提供一种研究胰高血糖素相关肽合成与分泌控制的方法。
