摘要
目的:探讨钙/钙调素依赖性蛋白激酶2β(Camk2b)低表达对1,4-苯醌(1,4-BQ)致K562细胞线粒体毒性的影响,初步揭示Camk2b对于细胞抗氧化损伤的作用和意义。方法:检测在1,4-BQ染毒后K562细胞Camk2bmRNA的表达,选择Camk2b抑制剂KN93的适宜浓度,通过荧光定量PCR法和Westernblot法验证Camk2bmRNA和蛋白表达,构建Camk2b低表达K562细胞。在0、10、20μmol/L 1,4-BQ染毒后,通过细胞增殖实验、活性氧实验、ATP生成量实验、线粒体膜电位实验、钙离子检测实验及细胞凋亡实验,检测Camk2b低表达K562细胞和对照细胞的增殖、活性氧、ATP、线粒体膜电位、凋亡率的改变。结果:1,4-BQ染毒后K562细胞Camk2bmRNA的表达下降。与对照细胞相比,Camk2b低表达细胞ATP生成量降低,线粒体膜电位降低,钙离子浓度升高,凋亡率上升(P均<0.01)。1,4-BQ染毒后,与相同浓度1,4-BQ染毒对照细胞相比,Camk2b低表达细胞ROS水平升高,ATP生成量降低,线粒体膜电位降低,钙离子浓度升高,凋亡率上升(P均<0.05或0.01)。结论:1,4-BQ染毒后,K562细胞Camk2bmRNA的表达降低。与对照细胞相比,Camk2b低表达细胞在1,4-BQ染毒后出现更明显的线粒体损伤,Camk2b对于细胞的抗氧化损伤具有重要意义。
OBJECTIVE:To investigate the effect from exposure to 1,4-benzoquinone (1,4-BQ) and of low expression of calcium/calmodulin dependent protein kinase 2 beta (Camk2b) on mitochondrial toxicity in K562 cells. METHODS:Expression of Camk2b mRNA in K562 cells was determined after 1,4-BQ exposure. Then, the appropriate concentrations of Camk2b inhibitor, KN93, was selected. The gene and protein expressions of Camk2b were verified by real-time PCR and Western blot,respectively,and the low expression of Camk2b was successfully constructed. After K562 cells were exposed to 0, 10, and 20 μmol/L 1,4-BQ, the following assays were conducted:cell proliferation,reactive oxygen species,ATP production,mitochondrial membrane potential, calcium ion and apoptosis. The same assays were conducted in K562 cells and control cells which showed low expression of Camk2b. RESULTS:Expression of Camk2b mRNA in K562 cells was reduced after 1,4-BQ exposure. Compared with the control cells, significant changes of the followings were detected: reduction of the ATP production of Camk2b low expression cells, reduction of the mitochondrial membrane potentials, increase of the calcium ion concentrations and increase of the apoptotic rates (all P< 0.01). After 1,4-BQ exposure and compared with the same concentrations of 1,4-BQ-treated control cells, significant changes were observed: increase of the ROS level of Camk2b low expression cells, reduction of ATP production, decrease of mitochondrial membrane potentials, increased of the calcium concentrations and increase of the apoptotic rates (P<0.05 or 0.01). CONCLUSION:After the K562 cells were exposed to 1,4-BQ,expression of Camk2b mRNA was reduced. Compared with the control cells,Camk2b low-expression cells showed more obvious mitochondrial damage after the 1,4-BQ exposure and Camk2b was apparently important for antioxidant damage to these cells.
作者
张虹
王彤
王坤
王博深
章梦莹
周燕华
浦跃朴
张娟
ZHANG Hong;WANG Tong;WANG Kun;WANG Boshen;ZHANG Mengying;ZHOU Yanhua;PU Yuepu;ZHANG Juan(Key Laboratory of Environmental Medicine, Engineering of Ministry of Education, School of Public Health, SoutheastUniversity, Nanjing 210009, Jiangsu, China)
出处
《癌变．畸变．突变》
CAS
2019年第4期261-267,共7页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金(81573120)
