CaMK-Ⅱ和PP2A在AlCl3致SH-SY5Y细胞tau蛋白异常磷酸化中的调控作用  

Regulatory role of CaMK-Ⅱand PP2A on hyperphosphorylated tau induced by AlCl3 in SH-SY5Y cells

作　　者：路小婷 崔双杰 张云玮 徐诗梦 巨晓芬 牛侨 Lu Xiaoting;Cui Shuangjie;Zhang Yunwei;Xu Shimeng;Ju Xiaofen;Niu Qiao(Department of Occupational Health,School of Public Health,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学公共卫生学院劳动卫生学教研室,太原030001

出　　处：《卫生研究》2020年第4期534-539,共6页Journal of Hygiene Research

基　　金：国家自然科学基金(No.81472959,81001241)。

摘　　要：目的探讨钙调蛋白激酶-Ⅱ(CaMK-Ⅱ)和蛋白磷酸酶-2A(PP2A)在AlCl3致人源性神经母细胞瘤细胞株(SH-SY5Y)细胞tau蛋白异常磷酸化中的调控作用。方法选取SH-SY5Y细胞进行AlCl3染毒和CaMK-Ⅱ拮抗剂KN93、PP2A激动剂鞘氨醇干预,实验分为对照组,AlCl3低、中、高染毒组(200、400和800μmol/L Al3+),KN93干预组(800μmol/L Al3++0.5μmol/L KN93)和鞘氨醇干预组(800μmol/L Al3++5 nmol/L鞘氨醇),染毒和干预48 h后,采用CCK-8法测定细胞活力,ELISA方法测定CaMK-Ⅱ、PP2A的含量,Western-Blot检测细胞总tau(tau5)及Thr-181、Thr-231、Ser-262、Ser-396磷酸化水平。结果细胞活力测定结果显示:与对照组相比,AlCl3中、高染毒组细胞活力降低(P<0.05);与AlCl3高染毒组相比,KN93干预组和鞘氨醇干预组细胞活力增高(P<0.05),且鞘氨醇干预组细胞活力与对照组相比差异无统计学意义。ELISA实验结果显示:与对照组相比,AlCl3低、中、高染毒组CaMK-Ⅱ含量增高(P<0.05),PP2A含量降低(P<0.05);与AlCl3高染毒组相比,KN93干预组和鞘氨醇干预组CaMK-Ⅱ含量降低(P<0.05),PP2A含量增高(P<0.05)。Western-Blot结果显示:与对照组相比,AlCl3低、中、高染毒组tau5和Thr-181、Thr-231、Ser-396磷酸化水平增高(P<0.05),AlCl3高染毒组Ser-262磷酸化水平增高(P<0.05);与AlCl3高染毒组相比,KN93干预组和鞘氨醇干预组tau5和Thr-181、Thr-231、Ser-262、Ser-396磷酸化水平降低(P<0.05),且KN93干预组Ser-262磷酸化水平,鞘氨醇干预组Thr-181、Thr-231、Ser-396磷酸化水平与对照组相比差异无统计学意义。结论AlCl3可引起SH-SY5Y细胞Thr-181、Thr-231、Ser262、Ser-396磷酸化水平异常增高,其机制与CaMK-Ⅱ和PP2A的变化有关;CaMK-Ⅱ主要调控Ser-262磷酸化水平,PP2A主要调控Thr-181、Thr-231、Ser-396磷酸化水平,提示AlCl3致SH-SY5Y细胞tau蛋白异常磷酸化与PP2A密切相关。OBJECTIVE To explore the role of calcium/calmodulin-dependent protein kinaseⅡ(CaMK-Ⅱ)and protein phosphatase-2A(PP2A)onhyperphosphorylation of tau induced by AlCl3 in SH-SY5Y cell.METHODS SH-SY5Y cells were assigned to the control group,AlCl3 low,middle,high exposed groups(200,400和800μmol/L Al3+),KN93 intervention group(800μmol/L Al3++0.5μmol/L KN93)and sphingosine intervention group(800μmol/L Al3++5 nmol/L sphingosine).After 48 h of exposure and intervention,the viabilities of cells were measured by CCK-8 assay,the contents of CaMK-Ⅱand PP2A were determined by ELISA,and the expression of tau5 and phosphorylation of Thr-181,Thr-231,Ser-262 and Ser-396 were detected by Western-Blot.RESULTS The viabilities of cells in AlCl3 middle and high exposed groups were significantly lower than that of the control group(P<0.05).Compared with the AlCl3 high exposed group,the viabilities of cells in KN93 intervention group and sphingosine intervention group were significantly increased(P<0.05),as well as the difference of the cell viability between sphingosine intervention group and the control group was not statistically significant.Compared with the control group,the contents of CaMK-Ⅱin AlCl3 low,middle,high exposed groups were significantly increased(P<0.05),while the contents of PP2A in those groups were significantly decreased(P<0.05).Compared with AlCl3 high exposed group,the contents of CaMK-Ⅱin KN93 intervention group and sphingosine intervention group were significantly decreased(P<0.05),and PP2A in those groups were significantly increased(P<0.05).The expression of tau5 and phosphorylation of Thr-181,Thr-231,Ser-396 in AlCl3 low,middle,high exposed group showed significantly higher than those of the control group(P<0.05),while the phosphorylation of Ser-262 in AlCl3 high exposed group showed significantly higher than that of the control group(P<0.05).After intervention with KN93 and sphingosine,the expression of tau5,and phosphorylation of Thr-181,Thr-231,Ser-262,Ser-396 in KN93 intervention group and

关 键 词：铝 磷酸化 tau 蛋白 钙调蛋白激酶-Ⅱ 蛋白磷酸酶-2A 

分 类 号：O614.31[理学—无机化学] Q593.2[理学—化学]