Pancreatic islet culture for islet transplantation

胰岛移植是替代胰岛素注射治疗糖尿病的一种新方法。胰岛细胞的体外培养是一个关键的过程，因为胰岛细胞在体外培养过程中有时会失去功能。在本研究中，我们重点研究了基质包膜，以改善培养，延长其胰岛素分泌。为了研究基质对胰岛细胞的影响，我们将胰岛细胞培养在包被多种基质的培养皿上，观察细胞脱离基质和胰岛素释放随葡萄糖水平的变化。从雄性Lewis大鼠中分离胰岛，然后用胰蛋白酶分散。将分散的细胞播撒在涂有I型胶原蛋白、IV型胶原蛋白、纤维连接蛋白、层粘连蛋白或不涂有层粘连蛋白的培养皿上。通过计数20次移液后的细胞数来测定细胞与基质的分离强度。在富糖培养基和贫糖培养基中进行葡萄糖刺激试验，用ELISA法测定培养上清中胰岛素的量。在I型胶原和层粘连蛋白上培养的胰岛细胞最容易分离，在IV型胶原上培养的细胞也容易分离，而在纤维连接蛋白和非包被塑料上培养的细胞则不容易分离。在层粘连蛋白上培养的细胞释放胰岛素量最多，刺激指数最高，而在纤维连接蛋白或无包被塑料上培养的细胞释放胰岛素量最少，刺激指数最低。这些结果提示，附着在培养皿上可以抑制胰岛细胞释放胰岛素，胰岛细胞应在易于分离的培养皿上培养。

Islet transplantation is a novel diabetic therapy instead of insulin injection. In vitro culture of islet is pivotal process, because islet cells sometimes lose their function during in vitro culture. In this study, we focused on matrices coating so as to improve the culture and prolong their insulin secretion. In order to investigate the effect of matrices, the islet cells were cultured on dishes coated with several matrices, and measured the detachment of the cells from them and insulin release in response to glucose level.The islets were isolated from male Lewis rats and then dispersed with trypsin. Dispersed cells were seeded on the dishes coated with type I collagen, type IV collagen, fibronectin, laminin or without. Intensity of detachment of the cells to the matrices was assayed by counting the detached cell number after 20 times pipetting. Glucose stimulation test was carried out in glucose-rich medium and glucose-poor medium, and the insulin amount in the culture supernatant was determined by ELISA. The islet cells cultured on type I collagen and laminin were most easily detached after pipetting, and cells on type IV collagen were also easily detached, but cells on fibronectin and noncoated plastic were failed to detach. Cells cultured on laminin released most amount of insulin and showed highest stimulation index, but the cells on fibronectin or noncoated plastic released the least insulin and showed the lowest stimulation index. These results imply that attachment on the culture dish could inhibit insulin release from islets cells and that islet cells should be cultured on easily detachable dish.