葡萄糖刺激下小鼠胰岛素分泌时相及胰岛素囊泡的分布变化

Phases of glucose-stimulated insulin secretion and changes of distribution of islet insulin secretory granules in mice

目的探讨葡萄糖刺激下小鼠胰岛素的动态分泌及胰岛素囊泡的分布变化。方法雄性C57BL小鼠50只,处死后分离小鼠胰岛,采用免疫荧光法孵育,镜下观察小鼠胰岛β细胞分布情况;取50个胰岛在含2.8mmol/L及16.7mmol/L葡萄糖的低糖和高糖KRBH缓冲液中灌流,采用放射免疫法分析胰岛素的动态分泌;另取60个胰岛分为低糖组与高糖组各30个,分别采用低糖和高糖KRBH缓冲液孵育5min,电镜下观察2组胰岛素分泌囊泡密度、分布及未成熟囊泡情况。结果小鼠胰岛β细胞位于胰岛中央;胰岛素第1时相分泌呈典型峰状,AUC为4.924±0.536,第2时相分泌呈平台样均匀分泌,AUC为11.180±0.690;低糖组胰岛素囊泡密度、距离β细胞膜〉50~300nm、〉300nm胰岛素分泌囊泡密度和未成熟胰岛素分泌囊泡密度（（213.3±10.2）、（41.4±1.96）、（152.4±9.52）、（2.9±0.21）个/0.1μm2）明显低于高糖组（（249.7±4.2）、（58.9±2.6）、（172.7±4.99）、（6.04±0.98）个/0.1μm2）（P〈0.05）,距离β细胞膜〈50nm胰岛素分泌囊泡密度（（19.6±0.7）个/0.1μm2）与高糖组（（18.1±0.94）个/0.1μm2）比较差异无统计学意义（P〉0.05）。结论葡萄糖刺激下的小鼠胰岛素第1时相分泌呈典型峰状,第2时相分泌呈平台样分泌;高糖刺激5min,胰岛素分泌囊泡合成增多,其中锚定囊泡分布无明显增多,但可释放池囊泡及储备池囊泡明显增多。

Objective To explore the glucose-stimulated dynamic insulin secretion and the distribution of islet insulin secretory granules in mice.Methods The pancreatic islets were isolated in 50 male C57BL mice,and immunofluorescence was used to identify the distribution of pancreaticβcells.Fifty islets were perfused with low-and high-glucose KRB buffer containing 2.8 mmol/L and 16.7 mmol/L glucose.The dynamic distribution of insulin was detected by radioimmunoassay.Another 60 islets were divided into low-glucose group and high-glucose group,with 30 islets in each group,using KRB buffer to incubate for 5minutes respectively.The density,distribution of insulin secretory granules and proportion of immature vesicle in cytoplasm were observed on electron microscope.Results The pancreaticβcells were located in the center of the islets.The first-phase insulin secretion showed typical peak-like secretion and AUCwas4.924±0.536.The second-phase insulin secretion showed platform-like secretion and AUC was 11.180±0.690.The density of insulin secretory granules,density of the granules with the distance of 50 to 300nm from membrance,density of the granules with the distance〉300nm from membrance,and density of immature vesicle were（213.3±10.2）,（41.4±1.96）,（152.4±9.52）,（2.9±0.21）/0.1μm2 in low-glucose group,significantly lower than those in highglucose group（（249.7±4.2）,（58.9±2.6）,（172.7±4.99）,（6.04±0.98）/0.1μm2）（P〈0.05）.There was no significant difference in the density of the granules with the distance〈50nm from membrance between low-glucose group（（19.6±0.7）/0.1μm2）and high-glucose group（（18.1±0.94）/0.1μm2）（P〉0.05）.Conclusion The first-phase insulin secretion shows typical peak shape,and the second-phase shows platform-like secretion.After high-glucose stimulation for 5 minutes,synthesis of insulin secretory granules increases,in which anchoring particles are not significantly increased,but granules of readily releasable pool and reserve pool increase significantly.