氯化镉引起线粒体分裂诱导小鼠精母细胞GC-2 spd凋亡  

Cadmium induces apoptosis of mouse spermatocytes(GC-2 spd) by promoting mitochondrial fission

作　　者：黄鼎宇 马兰 吕灵璐 胡恋 张玲 柳赟昊 Huang Dingyu;Ma Lan;Lyu Linglu;Hu Lian;Zhang Ling;Liu Yunhao(Department of Environmental Health and Occupational Medicine,School of Public Health,Wuhan University of Science and Technology,Wuhan 430065,China)

机构地区：[1]武汉科技大学医学院公共卫生学院环境卫生与职业医学系,武汉430065

出　　处：《中华劳动卫生职业病杂志》2022年第11期807-812,共6页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：湖北省高等学校优秀中青年科技创新团队计划(T2020003)。

摘　　要：目的探讨镉诱导小鼠精母细胞(GC-2 spd)凋亡的作用机制。方法于2021年3月,分别用5和10μmol/L氯化镉(CdCl_(2))染毒GC-2 spd细胞24 h,分别为CdCl_(2)5μmol/L染毒组(CdCl_(2)低剂量组)和CdCl_(2)10μmol/L染毒组(CdCl_(2)高剂量组),以未染毒的GC-2 spd细胞作为对照组。采用Mito-Track Red CMXRos线粒体荧光探针染色细胞检测线粒体形态,流式细胞术结合JC-1荧光探针检测线粒体膜电位的变化;使用细胞线粒体分离试剂盒和RIPA裂解液分别提取GC-2 spd细胞的线粒体蛋白、细胞浆蛋白和细胞总蛋白,Western blot检测线粒体稳态调节蛋白[分裂蛋白(FIS1)和融合蛋白(OPA1)]及细胞凋亡相关蛋白(Cytochrome c和cleaved Caspase-3)的表达。结果与对照组细胞比较,CdCl_(2)低剂量组和高剂量组细胞中JC-1红色/绿色荧光信号相对比值均明显降低(0.740±0.071、0.570±0.028),差异有统计学意义(P=0.017、0.004);线粒体形态由长管状变为点状,含点状线粒体的细胞比例明显升高(45.1%±3.7%和25.7%±4.9%),差异有统计学意义(P=0.005、0.001);CdCl_(2)低、高剂量组细胞中线粒体FIS1相对表达水平均明显升高(1.271±0.120、1.693±0.155),差异有统计学意义(P=0.046、0.000);OPA1相对表达水平明显降低(0.838±0.050、0.682±0.040),差异有统计学意义(P=0.049、0.001)。与对照组细胞比较,CdCl_(2)低剂量组细胞的胞浆中Cytochrome c蛋白相对表达水平(1.249±0.151)差异无统计学意义(P=0.075),然而CdCl_(2)高剂量组细胞胞浆中其相对表达水平明显升高(2.355±0.110),差异有统计学意义(P<0.001);CdCl_(2)低、高剂量组线粒体中Cytochrome c相对表达水平均明显降低(0.681±0.043、0.619±0.114),差异有统计学意义(P=0.004、0.001);细胞中cleaved Caspase-3蛋白水平升高(5.486±0.544、11.493±1.739),差异有统计学意义(P=0.004、<0.001)。结论CdCl_(2)可促进线粒体分裂,影响Cytochrome c蛋白在GC-2 spd细胞内分布并激活Caspase-3蛋白活性,引�Objective To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes(GC-2 spd).Methods In March 2021,GC-2 spd cells were exposed to different concentrations of CdCl_(2)for 24 hours,namely 5μmol/L CdCl_(2)(low-dose)group and 10μmol/L CdCl_(2)(high-dose)group,and unexposed GC-2 spd cells were used as control group.Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes.Mitochrondrial proteins,cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer,respectively.The expression of mitochondrial homeostasis regulatory proteins(FIS1 and OPA1),and apoptosis-related proteins(Cytochrome c and cleaved Caspase-3)were examined by Western blot.Results Compared with the cells in the control group,the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl_(2)groups decreased significantly(0.740±0.071,0.570±0.028),with a statistically significant difference(P=0.017,0.004);The morphology of mitochondria changed from long tube to point,and the proportion of cells containing point mitochondria increased significantly(45.1%±3.7%and 25.7%±4.9%),the difference was statistically significant(P=0.005,0.001);The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl_(2)groups was significantly higher(1.271±0.120,1.693±0.155),the difference was statistically significant(P=0.046,0.000);The relative expression level of OPA1 decreased significantly(0.838±0.050,0.682±0.040),and the difference was statistically significant(P=0.049,0.001).Compared with the control group,the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl_(2)was not significantly increased(1.249±0.151),and the difference was not statistically significant(P=0.075).How

关 键 词：镉 线粒体 精母细胞 细胞凋亡 细胞分裂 GC-2 spd细胞 线粒体形态 

分 类 号：R114[医药卫生—卫生毒理学]