慢性氟中毒大鼠大脑皮质神经细胞线粒体分布及其分裂基因表达改变

Alteration of mitochondrial distribution and gene expression of fission 1 protein in cortical neurons of rats with chronic fluorosis

目的探讨慢性氟中毒大鼠大脑皮质神经细胞胞体和突触内线粒体分布情况及线粒体分裂基因Fisl表达的改变。方法sD大鼠60只，雌雄各半，体质量100～120g，按完全随机设计法分为3组，每组20只。在饮水中加入不同剂量的氟（0、10和50mg／L）来饲养，实验期6个月。采用荧光素标记细胞色素C氧化酶Ⅳ亚型1抗体显示线粒体，观察线粒体在神经细胞胞体和突触内分布；用即时荧光定量PCR、Westernblot和免疫组织化学sP方法检测线粒体分裂基因Fisl表达。结果染氟组大鼠出现不同程度的氟斑牙，尿氟含量明显升高，表明成功地复制了慢性氟中毒大鼠模型。饲以10mg／L和50mg／L的染氟大鼠胞体内聚集型的神经细胞数[（34．8±4．7）和（39．3±3．0）]较对照组（4．4±2．3）显著增加（P〈0．05）；分裂基因FislmRNA转录水平[（3773±1292）和（1274±162）]较对照组（277±73）显著升高（P〈0．05）；Fisl蛋白免疫组织化学强阳性表达神经细胞数[（54．0±3．6）和（51．3±4．1）]和Westernblot法蛋白印迹平均吸光度值[（2．9±0．4）和（2．6±0．6）]水平较对照组[（25．2±2．5）和（1．8±0．2）]明显升高（P〈0．05）。结论摄人过量的氟可导致大鼠大脑皮质神经细胞线粒体在胞体和突触内重分布，诱导线粒体Fisl基因表达，促进线粒体分裂。神经细胞线粒体重分布可能与神经细胞Fisl高表达导致线粒体功能障碍有关。

Objective To investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 （ Fisl ） protein in the cortical neurons of rats with chronic fluorosis. Methods Sixty SD rats were divided into 3 groups （20 each ） according to weight hierarchy and fed with different concentrations of fluoride in drinking water （0, 10 and 50 mg/L, respectively ） for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COX IV and tubulin-ct were captured with fluorescence microscope. Fisl protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fisl mRNA was detected with real-time PCR. Results Varying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COX IV （34. 8 ± 4.7 and 39.3 ± 3.0, respectively） , and the expression of Fisl protein （ immunohistochemistry： 54. 0 ±3.6 and 51.3 ± 4. 1, respectively ; Western blot ： 2.9 ±0.4 and 2. 6 ± 0. 6, respectively） and mRNA （3773 ±1292 and 1274 ± 162, respectively） was markedly increased as compared with controls （4. 4 ± 2. 3, 25.2 ± 2. 5, 1.8 ± 0. 2 and 277 ± 73 ） over the experimental period of 6 months. Conclusions Excessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons （ i. e. , the increase in soma and the decrease in axon） , increased expression of Fisl gene and enhanced mitochondrial fission. The alteredmitochondrial distribution may be related to the high expression level of Fisl and a functional disorder of mitochondria.