检测线粒体通透性转换孔道评价胰岛β细胞线粒体功能

Evaluation of Mitochondrial Damage of Islet β Cells by Mitochondrial Permeability Transition Pore

目的评价高糖高脂作用下胰岛β细胞线粒体功能损害情况。方法分别于5.6 mmol/L和25mmol/L葡萄糖中加入0.4 mmol/L软脂酸,培养胰岛β细胞株INS-1细胞24 h,通过荧光染色,利用流式细胞技术和荧光显微镜检测线粒体通透性转换孔道(mPTP)、线粒体膜电位和活性氧(ROS)的改变,了解β细胞线粒体功能损害情况。以溴脱氧尿核苷掺入法测定细胞增殖情况,以Annexin V法测定细胞凋亡情况。结果在给予25mmol/L葡萄糖干预后,mPTP活性下降,线粒体膜电位升高,细胞增殖增加(均为P<0.05),ROS生成未见明显增多;给予25 mmol/L葡萄糖+0.4 mmol/L软脂酸后,mPTP活性和线粒体膜电位均下降,细胞凋亡增多、增殖减少(均为P<0.05),ROS生成增多。结论高糖作用使INS-1细胞mPTP活性下降、线粒体膜电位升高,提示细胞可能处于一种高代谢的不稳定状态;mPTP的检测有助于更全面地认识细胞糖毒性作用下的线粒体功能改变。

Objective To evaluate the mitochondrial damage of islet β cells under glucolipotoxicity by investigating the mitochondrial permeability transition pore（mPTP）.Methods Pancreatic β cell lines INS-1 cells were treated with 0.4 mmol/L palmitic acid and different concentrations of glucose（5.6 mmol/L or 25 mmol/L）.The mitochondrial membrane potential,mPTP and reactive oxygen species（ROS） were measured by flow cytometry and fluorescence staining technique to assess the mitochondrial damage.Cell proliferation was measured by 5-bromodeoxyuridine incorporation and cell apoptosis was detected by Annexin V method.Results Compared with the low glucose concentration,the high glucose concentration resulted in decreased mPTP activity（P〈0.05）,increased mitochondrial membrane potential（P〈0.05） and increased cell proliferation rate（P〈0.05）.There was no significant change in ROS generation.When cells were exposed to high glucose concentration and palmitic acid,both mPTP activity and mitochonhdrial membrane potential reduced（P〈0.05）,with increased cell apoptosis rate（P〈0.05） and increased ROS generation.Conclusion The high glucose concentration decreases mPTP and increases mitochondrial membrane potential,suggesting that cells may remain in an unstable high metabolic state.Evaluation of mPTP may contribute to a more comprehensive understanding of mitochondrial dysfunction under glucotoxictiy.