氟中毒对神经母细胞瘤细胞株尼古丁受体蛋白和基因表达的影响

目的 观察氟中毒对神经细胞中尼古丁受体亚单位蛋白和基因表达水平的影响及氟中毒引起尼古丁受体功能改变的发生机制。方法 体外培养人脑神经母细胞瘤细胞,在培养液中加入不同浓度的氟化物或加入抗氧化剂,培养48h后测定细胞3-4,5-二甲基噻唑-2,5-二酚基四唑溴化物(MTT)和蛋白氧化水平;用蛋白印记方法测定α3和α7尼古丁受体亚单位蛋白水平;用逆转录-聚合酶链反应方法测定尼古丁受体亚单位mRNA水平。结果 经氟处理的神经细胞中MTT水平降低,高浓度氟组比对照组低49％;蛋白氧化水平升高,高浓度氟组比对照组高72％;高浓度氟组神经细胞中α3和α7尼古丁受体亚单位蛋白含量分别比对照组减少51％和47％,但mRNA表达水平未见改变;用抗氧化剂处理可减弱氟中毒对尼古丁受体的损害。结论 氟中毒可引起神经母细胞瘤细胞受损,降低尼古丁受体蛋白水平,其机制与该受体的基因表达水平无关,但与自由基的损害有关。

Fluorosis on expression of nicotinic acetylcholine receptors in protein and gene levels in human SH-SY5Y neuroblastoma cells

OBJECTIVE: To investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR). RESULTS: In high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride. CONCLUSION: Fluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.