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目的建立外周血淋巴细胞线粒体呼吸链复合体Ⅰ活性的检测方法,观察T2DM患者线粒体呼吸链复合体工活性是否发生改变。方法收集T2DM患者47例(T2DM组)和健康体检者38名(NC组)临床资料和实验室检查结果。差速离心法分离线粒体,并利用透射电镜观察证实。反复冻融破线粒体膜,酶标仪检测氧化型葵基泛醌(DB)在272~247 nm波长处吸光度(OD)的变化。结果从外周血淋巴细胞中成功地提取了线粒体,线粒体蛋白质浓度为(0.03010±0.01589)mg/ml,272~247 nm波长的△OD在1 min内随着时间的延长呈增加趋势,且T2DM组复合体Ⅰ活性低于NC组[(16.51±5.45)vs(24.37±8.45)nmol/(min·mg),P=0.026]。结论成功建立检测外周血淋巴细胞线粒体呼吸链复合体Ⅰ活性动力学的方法。线粒体呼吸链复合体Ⅰ活性的改变可能与T2DM发病相关。
Objective To establish a method to detect the activity of mitochondrial respiratory chain complex Ⅰ in peripheral blood lymphocytes and observe whether the mitochondrial respiratory chain complex activity in T2DM patients is changed. Methods The clinical data and laboratory findings of 47 patients with T2DM (T2DM group) and 38 healthy controls (NC group) were collected. Mitochondria were isolated by differential centrifugation and confirmed by transmission electron microscopy. The mitochondrial membrane was frozen and thawed repeatedly, and the change of absorbance (OD) at the wavelength of 272 ~ 247 nm was detected by microplate reader. Results Mitochondria were successfully extracted from peripheral blood lymphocytes. The concentration of mitochondrial protein was (0.03010 ± 0.01589) mg / ml. The △ OD at 272 ~ 247 nm increased with the increase of time within 1 min. The activity of complex I was lower than that of NC group [(16.51 ± 5.45) vs (24.37 ± 8.45) nmol / (min · mg), P = 0.026]. Conclusion The method of detecting the kinetics of respiratory chain complex I in peripheral blood lymphocytes has been successfully established. Changes in the activity of mitochondrial respiratory chain complex I may be related to the pathogenesis of T2DM.