【摘要】 目的 探讨氟中毒对小鼠小胶质细胞株BV2和小鼠来源神经母细胞瘤细胞株N2a及小鼠大脑皮质神经细胞损伤及自噬的影响。方法 8周龄清洁级雄性C57BL/6J野生型小鼠，随机分为对照组(饮水含氟量<0.5 mg/L)、低氟组(饮水含氟量10.0 mg/L)和高氟组(饮水含氟量50.0 mg/L),每组10只喂养12周；BV2及N2a正常培养后分对照组、低氟组(培养基含氟量1.0 mmol/L)和高氟组(培养基含氟量1.5 mmol/L);采用CCK8检测不同浓度氟处理下BV2和N2a细胞增殖活力变化，氟离子选择电极法检测造模小鼠尿、血、骨氟含量变化；蛋白免疫印迹法(Western blot)检测BV2、小鼠大脑皮质中小胶质细胞活化标志物离子钙结合衔接分子1(Iba-1)的表达变化及N2a细胞、BV2细胞和小鼠大脑皮质中自噬相关微管相关蛋白1轻链3(LC3)及自噬受体蛋白P62蛋白(SQSTM1/P62)的表达水平；线粒体动力学及自噬融合分裂相关蛋白线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)、动力相关蛋白1(Drp1)、张力蛋白同源物诱导的假定激酶1(Pink1)及E3泛素连接酶(Parkin)的表达水平。结果 CCK8结果显示，1.5 mmol/LNaF处理的BV2、N2a细胞增殖活力明显下降(P<0.001,);低氟组小鼠氟斑牙发生率为60%,高氟组小鼠氟斑牙发生率为80%;与对照组比较，染氟组小鼠尿、血、骨氟含量明显上升(P<0.001);蛋白免疫印迹结果显示，与对照组比较，高氟组的小鼠大脑皮质和BV2的小胶质细胞活化标志物Iba-1表达水平显著升高(P<0.001,P<0.01),高氟组的小鼠大脑皮质和BV2及N2a细胞自噬相关蛋白LC3、Parkin、Pink1表达量显著增加(P<0.05)、P62水平显著下降(P<0.01);线粒体融合蛋白Mfn1、Mfn2水平显著增加(P<0.001),分裂蛋白Drp1无明显变化。结论 氟中毒可导致线粒体损伤、破坏线粒体融合分裂平衡并引起细胞自噬，为阐明氟中毒神经系统损伤与细胞自噬之间相互关系提供新思路。更多还原

【Abstract】 Objective To investigate the effect of fluorosis on microglia BV2 and N2a cells, and on the damages and autopany of cerebral cortical neurons of mice. Methods Eight-week-old clean-grademale mice of C57BL/6J wild-type were randomly divided intothe control group(fluoridecontent in drinking water was<0.5 mg/L), the low-fluoride group(fluoride in drinking water was 10.0 mg/L) andthehigh-fluoride group(fluoride in drinking water was 50.0 mg/L), All the mice(10 cases in each group)were fed for 12 weeks. BV2 and N2a cells were divided into the control group, thelow-fluoride group(medium fluoride 1.0 mmol/L) and the high-fluoride group(medium fluoride 1.5 mmol/L) after normal culture. CCK8 was used to detect thechanges in proliferative viability of BV2 and N2a cells under different concentrations of fluorine treatment. Fluoride ion-selective electrode method was used to detect the changes offluoride contentsin urine, blood and bone of the modeled mice. Protein immunoblotting was used to detect the changes of the expression of ion-calcium-binding bridging molecule-1(Iba-1), which served as a marker of microglia activation, in the cortex of the mouse. Western blot test(WB) was used to detect the expression levels of autophagy-related microtubule-associated protein 1 light chain 3(LC3), and autophagy receptor protein P62(SQSTM1/P62) in N2a cells, BV2 cells and mouse cerebral cortex, and further to detectmitochondrial dynamics and autophagy fusion and division-associated proteins Mitochondrial Fusion Protein 1(Mfn1), Mitochondrial Fusion Protein 2(Mfn2), and Dynamic related protein 1(Drp1), tension protein homologue-induced putative kinase 1(Pink1), and E3 ubiquitin ligase(E3 ubiquitin-protein ligase, Parkin) expression levels.Results CCK8 showed that the value-added viability of BV2 and N2a cells treated with 1.0 mmol/L and 1.5 mmol/L NaF decreasedsignificantly(P<0.01, P<0.0001); the incidence of dental fluorosis in mice in the low-fluoride group was 60%(6/10), and that in the high-fluoride group was 80%(8/10),whilethe fluoride contentinurine, blood, and bone of the high-fluoride group significantly increased compared with the control group(all P<0.001); protein immunoblotting was used to detect the changes ofthe expression of Iba-1 in BV2 and mouse cerebral cortex. LC3 and P62 in BV2, N2a cells and mouse cerebral cortex. Mfn1, Mfn2, Drp1, Pink1, and Parkin expression level changes in the expression level. Conclusion Fluorosis leads to mitochondrial damage, disrupt the balance of mitochondrial fusion and division and cause cellular autophagy, which provides a new way of thinking to elucidate the interrelationship between neurological damage and cellular autophagy in fluorosis.更多还原