NAC对Aβ25-35诱导SH-SY5Y细胞Tau蛋白过度磷酸化影响的研究  

Effect of NAC on Aβ25-35 induced over phosphorylation of Tau protein in SH-SY5Y cells

作　　者：曹帆帆[1] 张登海[1] 王莹[1] 彭彬[1] 张雪[1] 徐莉敏[2] 

机构地区：[1]第二军医大学附属公利医院中法合作中心实验室,上海200135 [2]第二军医大学附属公利医院检验科,上海200135

出　　处：《临床和实验医学杂志》2016年第20期1984-1987,共4页Journal of Clinical and Experimental Medicine

基　　金：上海市浦东新区卫生系统优秀青年医学人才培养计划(PWRq2013-11);上海市青年科技英才扬帆计划资助(15YF1410800)

摘　　要：目的探讨N-乙酰基半胱氨酸(NAC)对β淀粉样蛋白片段25-35(Aβ25-35)诱导SH-SY5Y细胞Tau蛋白过度磷酸化的影响。方法用凝聚态Aβ25-35刺激SH-SY5Y细胞,建立Tau蛋白过度磷酸化的细胞模型。分为空白组、对照组和试验组。空白组为未刺激的细胞,对照组给予20μmol·L^(-1)的Aβ25-35作用6 h,试验组先给予10 mmol·L^(-1)的NAC作用24 h,然后给予20μmol·L^(-1)的Aβ25-35共同作用6 h。通过蛋白免疫印迹法检测CDK 5的两个亚基CDK5和P35/P25的蛋白水平,以及Tau蛋白在Thr 212和Ser396位点的磷酸化水平;采用荧光酶标法检测氧自由基(ROS)。对三组细胞的CDK5和P35/P25的蛋白水平、Thr 212和Ser396位点的磷酸化水平和ROS进行比较。结果三组细胞ROS荧光值比较结果显示,空白组ROS荧光值为231±19,对照组为359±23,试验组为264±32。空白组最低,对照组最高,试验组ROS荧光值明显高于空白组(P<0.05),但低于对照组(P<0.05)。空白组Tau蛋白在T212和S396磷酸化水平分别为1.03±0.09、1.04±0.05,对照组分别为1.63±0.54、1.72±0.34,试验组分别为1.31±0.09、1.21±0.05,空白组最低,对照组最高,试验组细胞明显高于空白组(P<0.05),但低于对照组(P<0.05)。空白组CDK5、P35分别为1.00±0.06、1.00±0.18,对照组分别为0.75±0.14、1.12±0.13,试验组分别为0.19±0.21、0.99±0.09。空白组、对照组与试验组CDK5、P35相比较无显著差异(P>0.05)。空白组P25水平为1.10±0.16、对照组为1.69±0.20,试验组为1.19±0.03,空白组最低,对照组最高,试验组P25值低于对照组,P25值高于空白组,差异具有统计学意义(P<0.05)。结论 NAC可以降低Aβ25-35诱导SH-SY5Y细胞Tau蛋白磷酸化水平。Objective To investigate the effect of NAC on Aβ25 -35 induced over phosphorylation of Tau protein in SH - SY5Y cells. Methods The SH - SY5Y cells were stimulated by condensed A beta 25 - 35, and the cell model of Tan protein phosphorylation was established. They were divided into blank group, control group and experimental group. Blank group for unstimulated cells, while the control group was given 20 μmol/L A beta 25 - 35 at 6 h, the experimental group give first 10 mmol/L NAC for 24 h, then give 20 μmol/L interaction of beta amyloid 25 - 35 6 h. Protein immunoblot assay for detection of CDK 5 through the two subunits of CDK5 and P35/P25 protein levels, and tau protein in the phosphorylation of thr 212 and ser396 site ; using a fluorescence microplate method was used to detect the oxygen free radicals （ROS） and cells in the three groups of CDK5 and P35/P25 protein levels, the 212 and set396 site phosphorylation level and ROS were compared. Results The three groups of cells ROS fluorescence value comparison results showed that the ROS fluorescence value of the blank group was 231 ± 19, control group 359 ± 23, test group 264 ± 32. The lowest was in the blank group, and the highest was in the control group, the ROS fluorescence value in the experimental group was significantly higher than that in the blank group （ P 〈 0.05 ）, but lower than that of the control group （ P 〈 0.05 ）. The expression of Tan in the phosphorylation level of group T212 and S396 of the blank group, the control group and the test group were 1.03 ±0.09, 1. 04 ± 0.05 ; 1.63 ± 0.54, 1.72 ±0.34 and 1.31 ± 0.09, 1.21 ± 0.05 respectively. The lowest was in the blank group, the highest was in the control group and the level of T212 and S396 in the experimental group was significantly higher than that in the blank group （ P 〈 0.05 ）, but lower than that of the control group （ P 〈 0.05 ）. CDK5, P35 in blank group, control group and test group were 1 ±0.06, 1 ± 0.18; 0.75 ± 0.14, 1.12 ±0.13 and 0.19 ±0.21,0

关 键 词：NAC AΒ25-35 TAU蛋白 过度磷酸化 

分 类 号：R749.16[医药卫生—神经病学与精神病学]