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目的:探讨高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响及作用机制。方法:SD大鼠胰岛细胞原代培养,不同浓度的葡萄糖处理后,采用MTT比色法检测细胞存活率,Hoechst-PI染色观察细胞凋亡,电镜观察细胞超微结构改变,RT-PCR方法检测Bax及Bcl-2相关基因mRNA表达水平。结果:5.5 mmol·L~(-1),11.1 mmol·L~(-1)和22.2 mmol·L~(-1)葡萄糖处理后,胰岛细胞活性分别下降到78.08%±2.29%、58.39%±3.13%和36.05%±2.63%,与阴性对照组比较差异具有统计学意义(P<0.05);Hoechst-PI染色结果显示随着葡萄糖作用浓度的增加,凋亡细胞数量也增加;RT-PCR显示胰岛细胞经不同浓度葡萄糖处理后,Bax mRNA的表达明显上调,Bcl-2 mRNA表达下调(P<0.05);电镜观察结果显示随着葡萄糖作用浓度的增加,胰岛细胞超微结构损害程度依次加重。结论:高浓度葡萄糖能明显引起胰岛细胞活性的下降,诱导凋亡反应的发生,凋亡机制与Bcl-2家族蛋白相关。
Objective: To investigate the effect of high glucose on rat islet cell apoptosis in vitro and its mechanism. Methods: Primary cultured rat pancreatic islet cells were cultured in different concentrations of glucose. Cell viability was detected by MTT colorimetric assay. Apoptosis was observed by Hoechst-PI staining. Ultrastructural changes were observed by electron microscopy. Bax and Bcl-2 related gene mRNA expression levels. RESULTS: After treatment with 5.5 mmol·L -1, 11.1 mmol·L -1 and 22.2 mmol·L -1 glucose, the islet cell viability decreased to 78.08% ± 2.29% and 58.39% respectively 3.13% and 36.05% ± 2.63%, respectively, which were significantly different from those of the negative control group (P <0.05). The results of Hoechst-PI staining showed that the number of apoptotic cells increased with the increase of glucose concentration. The islet cells were treated with different concentrations of glucose, the expression of Bax mRNA was up-regulated and the expression of Bcl-2 mRNA was down-regulated (P <0.05). The electron microscopy results showed that the degree of ultrastructural damage of islet cells increased with the increase of glucose concentration. CONCLUSION: High concentration glucose can significantly decrease the activity of islet cells and induce the apoptosis reaction. The mechanism of apoptosis is related to Bcl-2 family protein.