Perfetti R, Henderson T E, Wang Y, Montrose-Rafizadeh C, Egan J M
Diabetes Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Pancreas. 1996 Jul;13(1):47-54. doi: 10.1097/00006676-199607000-00006.
Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.
大鼠胰岛原代培养物在长时间培养时会丧失葡萄糖反应性并最终死亡。在本研究中，我们评估了基质胶（一种基底膜提取物）对以下方面的影响：（i）胰岛细胞存活；（ii）葡萄糖刺激后的细胞反应性；以及（iii）胰岛素、胰高血糖素和生长抑素的mRNA水平。通过胶原酶消化分离胰腺胰岛，并将其接种于涂有或未涂有基质胶层的培养皿中。使用反向溶血空斑试验，我们确定了胰岛素分泌细胞的总数以及单个β细胞分泌的胰岛素量。在暴露于5 mM葡萄糖1小时后，与仅在无基质胶的情况下培养3天的新鲜分离胰岛相比，在基质胶上培养6周的6月龄大鼠胰岛的β细胞显示出相同数量的胰岛素分泌细胞（39.5±2.5%对37.1±2.6%）。此外，在基质胶上培养6周的细胞释放的胰岛素以葡萄糖依赖的方式增加（p<0.001），ED50为7 mM。然而，与从胰岛3天培养物中分离的β细胞释放的胰岛素相比，单个β细胞释放的胰岛素量减少了40 - 60%（p<0.02）。最后，在基质胶上培养6周的细胞中，胰岛素、胰高血糖素和生长抑素mRNA水平增加了35 - 55%（p<0.05）。这些数据表明基质胶对维持正常β细胞活性和功能具有营养作用，并可能为长期培养胰腺胰岛的新研究模型的开发铺平道路。
