Aoki M, Kagawa S, Yamamura T, Matsuoka A, Utsunomiya J
Second Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan.
Nihon Naibunpi Gakkai Zasshi. 1988 Feb 20;64(2):109-18. doi: 10.1507/endocrine1927.64.2_109.
In the present study, the culture system for preparing monolayer islet cells of the neonatal rat was applied and modified for use with the pancreas of the adult rat. In this procedure, whole pancreatic tissues were enzymatically dispersed and then cultured for 30 days in TCM 199 medium with either 5.5 mM glucose or 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose. Under culture conditions without 2-deoxy-2-fluoroglucose, the responsiveness of B cells was totally abolished by day 20 of culture. The addition of 2-deoxy-2-fluoroglucose destroyed fibroblasts selectively in a period of 20 days, yielding the monolayers mostly consisting of islet cells, and the morphological characteristics were well preserved at the end of the culture study period. After culture for 20 days in medium with 2-deoxy-2-fluoroglucose, insulin secretion was raised in a dose-dependent fashion due to the increasing concentrations of glucose, leucine and 2-ketoisocaproate. The dose-response curve for insulin secretion evoked by glucose was sigmoid with a Km of 7 mM glucose, and the secretion threshold was observed at a concentration of between 2.8 and 5.5 mM glucose. The ratios of the maximum level to the basal were 6, 5 and 3 respectively for glucose, leucine and 2-ketoisocaproate. The secretory competence was preserved in the B cells on day 30 as well. Addition of epinephrine or clonidine inhibited the glucose-induced insulin secretion dose-dependently. At a concentration of 10(-7) M, both drugs produced an 80% drop in insulin secretion evoked by glucose. The inhibitory effect of epinephrine or clonidine was reversed by 3 X 10(-5) M yohimbine or 10(-5) M phentolamine, whereas 10(-5) M propranolol had little or no effect and alpha adrenergic blockade of prazosin (5 X 10(-5) M) was weak as compared to that of yohimbine. At a high concentration (10(-5) M) of phenylephrine, a marked drop of insulin secretion was observed. In summary, the present culture system facilitates the establishment of monolayers of adult rat pancreas that consist mostly of islet cells. In addition, it is certain that the response of the B cells in 2-deoxy-2-fluoroglucose to nutrient secretagogues and the adrenergic modulation of insulin secretion are well preserved for a long-term culture period of 30 days. These preparations may provide a useful tool not only for the in vitro study of the B-cell function, but also for use in implantation resource.
在本研究中，应用并改良了用于制备新生大鼠单层胰岛细胞的培养系统，以用于成年大鼠的胰腺。在此过程中，将整个胰腺组织酶解分散，然后在含有5.5 mM葡萄糖或5.5 mM葡萄糖加1 mM 2-脱氧-2-氟葡萄糖的TCM 199培养基中培养30天。在没有2-脱氧-2-氟葡萄糖的培养条件下，培养至第20天时B细胞的反应性完全丧失。添加2-脱氧-2-氟葡萄糖在20天的时间内选择性地破坏了成纤维细胞，产生了主要由胰岛细胞组成的单层细胞，并且在培养研究期结束时形态特征得到了很好的保留。在含有2-脱氧-2-氟葡萄糖的培养基中培养20天后，由于葡萄糖、亮氨酸和2-酮异己酸浓度的增加，胰岛素分泌呈剂量依赖性增加。葡萄糖诱发的胰岛素分泌的剂量反应曲线呈S形，Km为7 mM葡萄糖，在2.8至5.5 mM葡萄糖的浓度下观察到分泌阈值。葡萄糖、亮氨酸和2-酮异己酸的最大水平与基础水平之比分别为6、5和3。第30天时B细胞的分泌能力也得以保留。添加肾上腺素或可乐定可剂量依赖性地抑制葡萄糖诱导的胰岛素分泌。在10^(-7) M的浓度下，两种药物均可使葡萄糖诱发的胰岛素分泌下降80%。肾上腺素或可乐定的抑制作用可被3×10^(-5) M育亨宾或10^(-5) M酚妥拉明逆转，而10^(-5) M普萘洛尔几乎没有作用，与育亨宾相比，哌唑嗪（5×10^(-5) M）的α肾上腺素能阻断作用较弱。在高浓度（10^(-5) M）的去氧肾上腺素作用下，观察到胰岛素分泌明显下降。总之，本培养系统有助于建立主要由胰岛细胞组成的成年大鼠胰腺单层细胞。此外，可以确定的是，在2-脱氧-2-氟葡萄糖存在的情况下，B细胞对营养促分泌剂的反应以及胰岛素分泌的肾上腺素能调节在30天的长期培养期内得到了很好的保留。这些制剂不仅可为B细胞功能的体外研究提供有用的工具，还可用于植入资源。
