镉诱导小鼠原代神经细胞程序性坏死的机制  

Mechanism of cadmium-induced necroptosis of mouse primary nerve cells

作　　者：何邵波 蒋传命 HE Shaobo;JIANG Chuanming(Department of Laboratory Medicine,Shaoyang University,Shaoyang,Hunan 422000,China)

机构地区：[1]邵阳学院医学检验学院,湖南邵阳422000

出　　处：《环境与职业医学》2021年第5期536-541,共6页Journal of Environmental and Occupational Medicine

摘　　要：[背景]镉是一种常见的重金属污染物,以往的研究主要聚焦其影响细胞自噬和凋亡,但其是否导致细胞程序性坏死尚不明确。[目的]研究镉诱导小鼠原代神经细胞程序性坏死的机制。[方法]以小鼠原代神经细胞为实验对象,10μmol·L^(-1)氯化镉分别处理细胞2、4、6、12、24、48 h,用台盼蓝染色法计算细胞存活率;采用流式细胞仪检测经10μmol·L^(-1)氯化镉处理后细胞周期的变化(24、48 h)及细胞程序性坏死水平(48 h);实时荧光定量PCR法检测经10μmol·L^(-1)氯化镉处理48 h后细胞程序性坏死相关基因如受体相互作用丝氨酸苏氨酸激酶3(RIPK3)、混合系列蛋白激酶样结构域蛋白(MLKL)的表达;蛋白印迹法检测5、10、20μmol·L^(-1)镉离子处理后自噬相关蛋白p62的蛋白表达水平;检测p62和自噬微管相关蛋白1轻链3-B(LC3-B)的荧光强度。[结果]小鼠原代神经细胞经10μmol·L^(-1)氯化镉处理4、6、12、24、48 h后细胞活力下降(P<0.05);相比较对照组(49.62%),细胞经10μmol·L^(-1)氯化镉处理24 h和48 h后分别有60.88%和82.94%的细胞停留在G0—G1期;10μmol·L^(-1)氯化镉处理48 h后,细胞程序性坏死水平(47.50%)高于对照组(0.01%),且细胞程序性坏死相关基因RIPK3和MLKL转录水平是对照组的6.9倍、3.7倍。经5、10、20μmol·L^(-1)氯化镉处理后小鼠原代神经元细胞自噬相关蛋白p62表达分别上调(587±17)%、(609±14)%、(893±16)%。经10μmol·L^(-1)氯化镉处理后细胞内自噬相关蛋白p62和LC3-B在细胞内共定位,且荧光强度高于对照组。[结论]镉离子能降低小鼠原代神经元细胞存活率,使细胞周期停滞在G0—G1期且提高细胞程序性坏死水平,可能与p62和LC3-B表达水平升高阻断细胞自噬流有关。[Background]Cadmium is a common heavy metal pollutant.Previous studies mainly focus on cadmium-induced autophagy and apoptosis,but whether it leads to cell necroptosis remains unclear.[Objective]This study investigates the mechanism of cadmium-induced necroptosis of mouse primary nerve cells.[Methods]Mouse primary neurons were treated with 10μmol·L^(-1) cadmium chloride for 2,4,6,12,24,and 48 h,respectively.The cell survival rate was calculated by Trypan blue staining.The changes of cell cycle(24 and 48 h)and the rate of cell necroptosis(48 h)after treatment with 10μmol·L^(-1) cadmium chloride were detected by flow cytometry.The expressions of genes related to cell necroptosis such as receptor interacting serine threonine kinase 3(RIPK3)and mixed lineage kinase domain-like protein(MLKL)after being treated with 10μmol·L^(-1) cadmium chloride for 48 h were measured by real-time fluorescence quantitative PCR.The protein expression of autophagy-related factor p62 and the fluorescence intensities of p62 and microtubule-associated protein 1 light 3-B(LC3-B)after 10μmol·L^(-1) cadmium chloride treatment were analyzed by Western blotting and fluorescence observation.[Results]The cell viability of mouse primary nerve cells were decreased after 4,6,12,24,and 48 h treatment with 10μmol·L^(-1) cadmium chloride(P<0.05).Compared with the control group(49.62%),60.88%and 82.94%of cells were arrested in the G0-G1 phase after treatment with 10μmol·L^(-1) cadmium chloride for 24 h and 48 h,respectively.The flow cytometry results showed that when being treated with 10μmol·L^(-1) cadmium chloride for 48 h,the rate of cell necroptosis was higher(47.5%)than that of the control group(0.01%),and the transcription of cell necroptosis related genes such as RIPK3 and MLKL were 6.9 and 3.7 times of the control group.The results of Western blotting showed that the expression of autophagyrelated protein p62 in mouse primary neurons were upregulated by(587±17)%,(609±14)%,and(893±16)%when being treated with 5,10,and 20μmol·L^(

关 键 词：镉 小鼠原代神经细胞 自噬 细胞程序性坏死 

分 类 号：R114[医药卫生—卫生毒理学]