Davalli A M, Bertuzzi F, Socci C, Scaglia L, Gavazzi F, Freschi M, DiCarlo V, Pontiroli A E, Pozza G
Istituto Scientifico San Raffaele, Cattedra di Clinica, Università di Milano, Italy.
Transplantation. 1993 Jul;56(1):148-54. doi: 10.1097/00007890-199307000-00028.
In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions. Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer. In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release. At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells. After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI 1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics). During this period, insulin release was 11.4 +/- 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2 +/- 2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8 +/- 0.2 (P < 0.001 vs. all the other groups), and 9.0 +/- 0.6 and 8.4 +/- 0.9 pg/islet/min in islets cultured in RPMI 1640 (without glucose), Ham's F12, and medium 199, respectively. After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L; static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42 +/- 0.06 to 0.60 +/- 0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24 +/- 0.06 to 0.48 +/- 0.06 pg/islet/min (P < 0.001) in islets cultured in medium 199. During perifusions, the paradoxical insulin release in response to an acute fall in glucose concentration disappeared, but a significant increase in response to high (16.7 mmol/L) glucose was observed only in islets previously cultured in medium 199. To assess the possible role of glucagon and of cAMP, additional perifusions were done in islets cultured for 48 hr in CMRL 1066 in the presence of glucagon (10 mumol/L) and IBMX (10 mumol/L); glucagon and IBMX were unable to modify the insulin response to 16.7 mmol/L glucose.(ABSTRACT TRUNCATED AT 400 WORDS)
在本研究中，对成年猪新鲜和培养的胰岛在静态（孵育）和动态（灌流）条件下的葡萄糖体外反应性进行了测试。通过自动化方法从24月龄动物的胰腺中分离胰岛，并在CMRL 1066、10%胎牛血清加抗生素中过夜培养；过夜培养后立即进行灌流的胰岛，在暴露于急性葡萄糖刺激（16.7 mmol/L）时胰岛素释放出现反常下降，而当向灌流缓冲液中加入异丁基甲基黄嘌呤（IBMX）时对急性葡萄糖有正常反应。此外，灌流液中葡萄糖浓度的急性降低引发了反常的胰岛素释放。在显微镜下，过夜培养后的胰岛显得松散且形状不规则；免疫组织化学显示外周A细胞和其他被膜细胞丢失。过夜培养后，将胰岛分为5组，并在不同的组织培养基中再培养48小时：CMRL 1066；RPMI 1640（无葡萄糖）；RPMI 1640（加11.1 mmol/L葡萄糖）；Ham's F12；以及培养基199（所有培养基均补充10%胎牛血清和抗生素）。在此期间，在CMRL 1066中培养的胰岛胰岛素释放为11.4±1.1 pg/胰岛/分钟，在RPMI 1640（11.1 mmol/L葡萄糖）中培养的胰岛为16.2±2.4，在RPMI 1640（无葡萄糖）、Ham's F12和培养基199中培养的胰岛分别为1.8±0.2（与所有其他组相比P<0.001）、9.0±0.6和8.4±0.9 pg/胰岛/分钟。在不同培养基中培养48小时后，评估胰岛对急性葡萄糖刺激（16.7 mmol/L；静态孵育）的反应性：在CMRL 1066和RPMI 1640（有和无葡萄糖）中培养的胰岛对急性葡萄糖刺激无胰岛素反应；相反，在Ham's F12中培养的胰岛胰岛素释放从0.42±0.06升至0.60±0.12 pg/胰岛/分钟（无显著性差异），在培养基199中培养的胰岛从0.24±0.06升至0.48±0.06 pg/胰岛/分钟（P<0.001）。在灌流过程中，对葡萄糖浓度急性下降的反常胰岛素释放消失，但仅在先前在培养基199中培养的胰岛中观察到对高（16.7 mmol/L）葡萄糖反应的显著增加。为评估胰高血糖素和cAMP的可能作用，在存在胰高血糖素（10 μmol/L）和IBMX（10 μmol/L）的情况下，对在CMRL 1066中培养48小时的胰岛进行了额外的灌流；胰高血糖素和IBMX无法改变对16.7 mmol/L葡萄糖的胰岛素反应。（摘要截断于400字）
