褪黑素对花萼海绵诱癌素诱导的tau蛋白过度磷酸化.doc

褪黑素对花萼海绵诱癌素诱导的tau蛋白过度磷酸化.doc褪黑素对花萼海绵诱癌素诱导的tau蛋白过度磷酸化
【摘要】目的探讨褪黑素(Mel)对花萼海绵诱癌素(CA)在成神经瘤细胞诱导的tau蛋白过度磷酸化的影响及其机制。方法采用鼠野生型成神经瘤细胞(N2aol/L Mel处理,用免疫荧光检测tau蛋白磷酸化水平,32P特异底物标记技术检测GSK3活性,免疫印迹技术检测PSer9GSK3β和GSK3β的表达水平,计算PSer9GSK3β/GSK3β的比率。结果①CA可在N2aelatonin on calyculin Ainduced tau hyperphosphorylation
ABSTRACT: Objective To investigate the in vivo effect of melatonin (Mel) on calyculin A(CA)induced tau hyperphosphorylation in neuroblastoma cells (N2aol/L Mel, detected the level of tau phosphorylation munofluorescence, and assayed the activities of GSK3 and the ratio of GSK3β phosphorylated at Ser9 site to total GSK3β. Results CA treatment led to tau hyperphosphorylation acpanied ultaneously ol/L Mel, the CAinduced tau hyperphosphorylation, GSK3 activation and the ratio of GSK3β phosphorylated at Ser9 site to total GSK3β decrease a cells from CAinduced tau hyperphosphorylation. Its protection may be related to the regulation of GSK3 activity and the ratio of GSK3β phosphorylated at Ser9 site to total GSK3β increase.
KEY el)处理,则可对抗CA引起的上述变化[3]。糖原合酶激酶3(GSK3)是可使tau的多个位点发生磷酸化的蛋白激酶[4]。根据报道GSK3激活参与氧化应激诱导的tau蛋白磷酸化[5],而另据报道,CA可引起氧化应激[6],因此,我们推测CA处理诱导神经细丝磷酸化可能也与GSK3激活有关,而抗氧化剂褪黑素也可能调节GSK3活性。为此,我们分别用CA处理、Mel和CA同时处理N2aa公司产品;过硫酸铵(AP)、二喹啉甲酸(BCA)蛋白检测试剂盒购自美国Pierce公司;***纤维素膜(NC膜)为 Hybond公司产品;单克隆抗体PHF1(识别Ser396/404位点磷酸化的tau,1∶300)、Tau1(识别非磷酸化的tau,1∶500)、多克隆抗体111e(识别总tau,1∶500)购自Sternberger公司;大鼠多克隆抗体