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目的研究哺乳期BDE-153染毒对成年大鼠海马线粒体和内质网结构和功能的影响,寻找BDE-153神经毒作用的靶细胞器,为研究BDE-153的神经毒性提供依据。方法选择出生4 d的新生雄性乳鼠40只,按体重随机分成:溶剂对照组(Olive oil),低剂量(1 mg/kg BDE-153)组,中剂量(5 mg/kg BDE-153)组和高剂量(10 mg/kg BDE-153)组,每组10只。仔鼠出生第10天染毒,按0.1 ml/10 g体重1次腹腔注射染毒BDE-153溶液或者橄榄油(仅对照组)。继续饲养两月后处死大鼠,在冰皿上迅速分离大脑皮质和海马,用流式细胞技术检测线粒体膜电位的改变,试剂盒检测线粒体Ca2+-Mg2+-ATP ase活力的改变,电子显微镜下观察线粒体和内质网超微结构的变化,用实时荧光定量PCR技术检测内质网跨膜蛋白132a(Transmembrane protein 132a,TMEM 132a)和Caspase-12基因的表达,用Western-Blot法测定TMEM 132a和原型Caspase-12(Procaspase-12),活化的Caspase-12(Cleaved caspase-12)蛋白的表达量。结果与对照组相比,各染毒组海马线粒体的平均荧光强度及Ca2+-Mg2+-ATP ase的活力变化不明显,差异无统计学意义(P>0.05),随着染毒剂量的增加,线粒体结构未见明显改变,内质网出现肿胀、扩张、变形,甚至溶解消失的现象。蛋白与基因的检测结果显示,与对照组比较,各染毒组TMEM132a基因和蛋白的表达量随着染毒剂量的增加显著降低,Caspase-12基因表达量升高明显,与此同时Procaspase-12蛋白表达量明显降低,而Cleaved caspase-12的蛋白表达量明显增加。结论内质网是BDE-153神经毒性的主要靶细胞器,可能通过Tmem132a和caspase-12信号分子介导其神经毒性。
Objective To investigate the effect of BDE-153 exposure on the structure and function of mitochondria and endoplasmic reticulum in hippocampus of adult rats during lactation, and to find the target organelles of BDE-153 neurotoxicity in order to study the neurotoxicity of BDE-153. Methods Forty neonatal male rats born 4 days old were randomly divided into four groups according to body weight: Olive oil, low dose (1 mg / kg BDE-153) and middle dose (5 mg / kg BDE-153) Group and high dose (10 mg / kg BDE-153) group, 10 rats in each group. The pups were exposed to BDE-153 solution or olive oil (intraperitoneal injection only) on day 10 of birth, and injected intraperitoneally with 0.1 ml / 10 g body weight (control group only). Rats were sacrificed two months later and the cerebral cortex and hippocampus were quickly separated from the ice dish. The change of mitochondrial membrane potential was detected by flow cytometry. The activity of mitochondrial Ca2 + -Mg2 + -ATP ase was detected by the kit and observed under an electron microscope The changes of ultrastructure of mitochondria and endoplasmic reticulum, the expression of transmembrane protein 132a (TMEM 132a) and Caspase-12 gene were detected by real-time fluorescence quantitative PCR. The expression of TMEM 132a and Prototype Caspase-12 (Procaspase-12), activated Caspase-12 (Cleaved caspase-12) protein expression. Results Compared with the control group, the mean fluorescence intensity of mitochondria and the activity of Ca2 + -Mg2 + -ATPase in the hippocampus were not changed significantly (P> 0.05), and the mitochondria No significant changes in the structure, endoplasmic reticulum swelling, expansion, deformation, and even dissolved disappear phenomenon. The results of protein and gene analysis showed that compared with the control group, the expression of TMEM132a gene and protein in each exposure group significantly decreased with the increase of exposure dose and the expression of Caspase-12 increased significantly. Meanwhile, Procaspase-12 Protein expression was significantly reduced, while Cleaved caspase-12 protein expression increased significantly. Conclusion The endoplasmic reticulum (ER) is the main target organelle of BDE-153 neurotoxicity, which may mediate its neurotoxicity through Tmem132a and caspase-12 signaling molecules.