游离脂肪酸对大鼠胰岛分泌功能和胰岛细胞凋亡的影响研究

EFFECT OF FREE FATTY ACID ON ISOLATED ISLET CELLS FUNCTION AND APOPTOSIS

目的:研究游离脂肪酸对体外培养SD大鼠胰岛功能和胰岛细胞凋亡的影响并探讨可能机制。方法:取健康雄性SD大鼠胰岛原代分离培养,分为6组,培养1d:NC1组(5.6mmol/L葡萄糖)、FFA1组(0.25mmol/L)、FFA2组(0.5mmol/L);培养3d:NC2组(5.6mmol/L葡萄糖)、FFA3组(0.25mmol/L)、FFA4组(0.5mmol/L);每组6个样本,每个样本20个胰岛。采用RT-PCR扩增胰岛素、PDX-1、Bax、Caspase-3基因mRNA表达,TUNEL法检测胰岛细胞凋亡率,放射免疫法检测胰岛素水平。结果:①FFA1、FFA2组Insulin、PDX-1基因表达量、胰岛素分泌较NC1组明显下降,FFA1、FFA2组凋亡基因Bax、Caspase-3表达量及胰岛细胞凋亡率较NC1组明显升高,差异有统计学意义(P<0.05);②FFA3、FFA4组Insulin、PDX-1基因表达量、胰岛素分泌较NC2组明显下降,FFA3、FFA4组凋亡基因Bax、Caspase-3表达量及胰岛细胞凋亡率较NC2组明显升高,差异有统计学意义(P<0.05)。结论:游离脂肪酸抑制体外培养SD大鼠胰岛细胞的胰岛素分泌功能,其可能机制是FFA抑制胰岛素基因、PDX-1基因的合成,刺激胰岛细胞凋亡基因Bax、Caspase-3表达,并导致胰岛细胞凋亡,最终导致胰岛细胞分泌功能下降。

Objective：To investigate the effect of free fatty acid （FFA） on the functions as well as apoptosis of islet cells ex vivo and its mechanisms in SD rats. Methods： Based on the concentration of FFA and treat- ment duration, islets isolated from SD rats were divided into six groups： normal control group 1 （NC1, 1- day treatment with 5.6 mmol/L glucose alone）, FFA group 1 （FFA1, 1-day treatment with the combina- tion of 5.6 mmol/L glucose and 0.25 mmol/L FFA）, FFA group 2 （FFA2, 1-day treatment with the com- bination of 5.6 mmol/L glucose and 0.5 mmol/L FFA）, normal control group 2 （NC2, 3-day treatment with 5. 6 mmol/L glucose alone）, FFA group 3 （FFA3, 3-day treatment with the combination of 5. 6 mmol/L glucose and 0.25 mmol/L FFA）, and FFA group 4 （1-day treatment with the combination of 5.6 mmol/L glucose and 0.5 mmol/L FFA）. Each group contained 6 samples which consisted of 20 islets. At time point required, the expression level of insulin, PDX-1, Caspase-3, and Bax genes were detected with reverse transcription-polymerase chain reaction （RT-PCR）. The apoptotic index of islet cells was evaluated by TUNEL assay. Radioimmunoassay was used to measure the concentration of insulin secreted by islet cells after the exposure to exogenous glucose at various concentrations in vitro. Results： A higher levels of Caspase-3 and Bax transcripts but not insulin and PDX combination of 5.6 mmol/L glucose and o. 25 mmol/L 1 were detected in the islet ceils treated with the FFA （group FFA1） or 0.5 mmol/L FFA （group FFA2） for one day compared to those treated with glucose alone （NC1, P 〈0. 05）. Similar patterns were observed in the islet cells treated with the same conditions for three days （groups FFA3 and FFA4）. There was a dramatic insulin secretion and reduction in glucose-stimulated a lower level of apoptotic index in the islet cells treated with the combined treatment of glucose and FFA （FFA1-FFA4） relative to those treated with glucose alone （NC1 and NC2, P 〈0.05）. Conclusion： Taken together, FFA exerts its inhibi- tion on glucose-simulated insulin secretion by islet cells probably through its role in the suppression of insu- lin and PDX-1 expression that upregulate the expression of pro-apoptotic genes, Bax and Caspase-3 leading to islet cell apoptosis and dysfunction.