原代分离的大鼠胰岛细胞对葡萄糖刺激胰岛素分泌的反应性研究

Study on the Glucose-Induced Secretion of Insulin in Isolated Rat Islets

目的:研究原代分离的大鼠胰岛对葡萄糖刺激的胰岛素分泌反应性。方法:胶原酶原位灌注法分离大鼠胰岛,在含0.5%BSA、5.5或11.1mmol/L葡萄糖的培养基中培养不同时间后,用含0.2%BSA、3.3mmol/L葡萄糖的KRB缓冲液预培养胰岛30min,分别换入含不同浓度葡萄糖KRB缓冲液,培养1h,收集上清,RIA法测定胰岛素浓度。结果:大鼠胰岛过夜培养后,在基础(3.3mmol/L)和高浓度(16.7mmol/L)葡萄糖条件下胰岛素分泌量分别为(12.4±3.2)和(45.2±4.2)μU/ml/10islets/h;5.5mmol/L和11.1mmol/L葡萄糖浓度下培养12h和20h后,胰岛对葡萄糖的反应性均明显高于16.7mmol/L和22.5mmol/L葡萄糖组(P<0.05);体外培养5d后,对高糖的反应性为(4.28±0.67)倍。结论:原代分离的大鼠胰岛可在(1～5)d内保持对葡萄糖的反应性。

Objective To study the insulin secretion responsiveness induced by glucose stimulation in isolated rat islets. Methods Rat islets were isolated after in situ eollagenase digestion through pancreatic duet perfusion. The islets were usually cultured overnight （ 16 - 20h） in RPMI 1640 media with 0.5% BSA and 11. lmmol/L glucose. The test began with a pre - culture of the islets for 30min in KRB buffer with 0.2% BSA, 3.3mmol/L glucose, then the amounts of insulin produced after exposure to different strengths of glucose （3.3,5.6,8.7,11.1,16.7 and 22.5mmol/L） in KRB barfer for lh were assayed. Islets were also cultured overnight in DMEM media with different strengths of glucose to examine the possible toxic injury with high concentrations of glucose （ 16.7 and 22.5mmol/L）. For viability test of the islets （ lwk or longer）, they were cultured in DMEM media with 5.5mmol/L glucose. Resuits The amounts of insulin produced after exposure to KRB buffer for lh with basal （3.3mmol/L） and high （16.7mmol/L） strength of glucose were （ 12.4 ± 3.2） and （45.2 ± 4.2）μU/ml/islets/h respectively. Islets cultured overnight in media with low concentration （5.5 or 11.1mmol/L） glucose secreted significantly more insulin than those cultured in media with higher concentration （16.7 and 22.5mmol/L） glucose （P 〈0.05 ）. After cultured for 5 days, the islets retained excellent response to glucose stimulation with increased insulin secretion （4.28 ± 0.67 fold of basal output of insulin in culture with high concentration of glucose）. Conclusion Isolated rat islets could retain high responsiveness to glucose stimulation for 1 ～5 days under optimal culture condition.