氟对中枢神经系统钙/钙调素依赖性蛋白激酶Ⅱ α亚单位mRNA和蛋白表达的影响

目的 观察氟对人神经母细胞瘤细胞(SH-SY5Y细胞)及发育期大鼠海马钙/钙调素依赖性蛋白激酶Ⅱα亚单位(α-CaMKⅡ)mRNA及蛋白表达水平的影响.方法 ①离体细胞实验:体外培养SH-SY5Y细胞,在培养液中加入NaF,使培养液中氟离子(F-)终浓度分别为0(对照)、0.05、0.50、2.00、5.00 mmol/L.培养48 h后,用qRT-PCR和Western blot技术检测细胞α-caMKⅡmRNA和蛋白水平.②动物实验:12只SD大鼠按雌∶雄=3∶1自然交配,待仔鼠出生后1 d,仔鼠及其哺乳母鼠-并按体质量随机分为3组.哺乳母鼠分别饮用含F-为0(对照)、2、3 mmol/L的蒸馏水,仔鼠通过乳汁摄入氟.每组有8只仔鼠在出生第14天处死,应用Western blot技术检测海马α-CaMKⅡ蛋白水平;另8只仔鼠自出生后21 d起,直接饮用与其哺乳母鼠相同染氟剂量的水溶液,至第28天处死.检测海马α-CaMKⅡ蛋白水平.结果 ①离体细胞实验:随着染氟剂量的增加,SH-SY5Y细胞中α-CaMKⅡ mRNA和蛋白水平依次降低.0、0.05、0.50、2.00、5.00 mmol/L组SH-SY5Y细胞中α-CaMKⅡ mRNA水平分别为1.00±0.00、0.77±0.18、0.40±0.11、0.22±0.06、0.15±0.03,蛋白水平分别为100.00±0.00、76.17±2.08、59.16±2.12、48.52±2.71、43.51±2.57,任意两组比较,差异有统计学意义(P<0.05或<0.01).②动物实验:2、3 mnlo/L组出生第14天和第28天仔鼠海马α-CaMKⅡ蛋白水平(75.02±2.88、73.83±3.88和81.00±2.54、45.70±2.34)低于对照组(100.00±0.00、100.00±0.00,P均<0.01),3 mmol/L组出生第28天仔鼠海马α-CaMKⅡ蛋白水平低于2 mmol/L组(P<0.01).结论 氟能降低神经细胞及海马中α-CaMKⅡ mRNA和蛋白表达水平,这可能是氟中毒引起学习记忆损害的机制之一.

Abstract：

Objective To investigate the effect of fluoride on the expression of a subunit of calcium/calmodulin-dependent protein kinase- Ⅱ (α-CaMK Ⅱ ) at both mRNA and protein levels in human neuroblastoma cells were cultured in DMEM with final concentrations of NaF 0(control) ,0.05,0.50,2.00,5.00 mmol/L, respectively, for 48 hours. Then quantitative RT-PCR and Western blot were performed to detect the expression level of α-CaMK Ⅱ P1 (postnatal day 1) pups together with their mothers were randomly divided into three groups. Lactating rats were given drinking water containing NaF at concentrations 0(control) ,2,3 mmol/L. And pups were exposed to NaF through milk. In each group, 8 pups were sacrificed on day 14 after birth. In post-weaning period, another 8 pups in each group were given drinking water with the same dose of fluoride as their mother's 21 day after birth. After then, these pups were killed on day 28, and hippocampus was dissected immediately and Western blot was conducted mRNA and protein levels were decreased. When NaF concentrations were 0,0.05,0.50,2.00,5.00 mmol/L, the mRNA relative ratios of α-CaMKⅡ in SY5Y cells were 1.00 ± 0.00,0.77 ± 0.18,0.40 ± 0.11,0.22 ± 0.06 and 0.15 ± 0.03, and protein levels of α-CaMK Ⅱ were 100.00 ± 0.00,76.17 ± 2.08,59.16 ± 2.12,48.52 ± 2.71 and 43.51 ± 2.57, any mmol/L group, hippocampus α-CaMK Ⅱ protein levels on day 14 and 28(75.02 ± 2.88,73.83 ± 3.88 and 81.00 ± 2.54,45.70 ± 2.34) were significantly lower than that of control groups(100.00 ± 0.00,100.00 ± 0.00, all P < 0.01). In 3 mmol/L group, hippocampus α-CaMKⅡ protein level on day 28 was lower than that of 2 mmol/L group (P < 0.01). Conclusion Fluoride can decrease mRNA and protein levels of α-CaMK Ⅱ in nerve cells and hippocampus, which may be one of the mechanisms of learning and memory impairment by fluorosis.