高糖对小鼠胰岛和胰岛β细胞株INS-1E的长期作用

Chronic effects of high glucose concentration on function of isolated mouse islets and islet β cell line INS-1E

目的探讨高糖对胰岛B细胞INS-1E和小鼠胰岛的慢性作用。方法雌性NMRI小风6-10周龄，苯巴比妥腹腔注射麻醉，应用胶原酶技术消化胰腺分离胰岛。传代培养的INS-1E细胞和分离的小鼠胰岛分别于含11．1，25．0mmol/L葡萄糖的RPMll640培养液中培养72h，然后于含3．3，16．7mmol／L葡萄糖的Krebs-Ringer缓冲液中培养60min，留取上清液行胰岛素测定。INS-1E细胞在含不同浓度的葡萄糖的RPMI1640培养液中培养72h，提取其总RNA，合成相应的cDNA，再行RT-PCR检测胰十二指肠同源异形盒-1（Pdxl），胰岛素1（Insl），胰岛素2（Ins2）和葡萄糖转运子2（Glut2）的基因表达。结果高糖培养后INS-1E细胞和小鼠胰岛的基础INS分泌增加[INS-1E细胞：（10．47±0．78）vs（7．71±0．59）ng／10000细胞，P〈0．01；小鼠胰岛：（3．85±0．26）vs（2．18±0．21）μg／L，P〈0．001]，糖刺激的INS分泌减少[INS-1E细胞：（17．11±1．98）vs（30．76±2．20）ng／10000细胞，P〈0．001；小鼠胰岛：（14．78±1．03）VS（20．46±1．49）μg／L，P〈0．01]；高糖处理后INS-1E细胞的Pdxl，Insl，Ins2和Glut2的mRNA水平下降。结论高糖对胰岛β细胞具有慢性毒性作用。

Objective To explore the chronic effects of excess glucose on INS-1E cells and isolated mouse islets. Methods Islets were isolated by the collagenase digestion from adult female NMRI mice （6-10 weeks） after induction of anesthesia by phenpbarbital. INS-1E cells and isolated mouse islets were cultured in RPMI 1640 supplemented with 11.1 or 25.0mmol/L glucose for 72h, respectively. Then the medium were changed to Krebs-Ringer buffer containing 3.3 or 16.7mmol/L glucose and incubated for 1 h. Correspondingly upper medium was collected for insulin assay. Total RNA was extracted from INS-1E cells after 72h incubation with different concentration of glucose, then cDNA was synthesized and mRNA expressions of Pdxl, insulin 1, insulin 2, and GLUT2 were determined by RT-PCR. Results After incubation with excess glucose, basal insulin secretion was increased in both INS-1E ceils and isolated mouse islets [INS-1E ceils： （10.47± 0.78） vs （7.71 ± 0.59） ng/10000 cells, P 〈 0.01; isolated mouse islets： （3.85 ±0.26） vs （2.18± 0.21） μg/L, P 〈 0.001], and glucose-stimulated insulin secretion was impaired [INS-1E cells： （17.11 ± 1.98） vs （30.76 ± 2.20） ng/10000 ceils, P 〈 0.001; isolated mouse islets： （14.78 ± 1.03） vs （20.46± 1.49）μg/L, P 〈 0.01]. Pdxl, insulin 1, insulin 2, and GLUT2 mRNA levels were reduced in INS-1E cells after treatment of excess glucose. Conclusion Long-term exposure to supraphysiologic glucose concentrations may cause dysfunction of pancreatic islet cells.