硒、锌干预对体外淀粉样前体蛋白酶解通路的影响

INTERVENTION OF SELENIUM AND ZINC ON AMYLOID PRECURSOR PROTEIN ZYMOLYTIC PATHWAY IN VITRO

目的研究硒、锌干预对体外淀粉样前体蛋白(amyloid precursor protein,APP)酶解通路的影响。方法用0.1μmol/L的硒、锌处理胚鼠皮层神经元细胞,采用酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测γ-内切酶活性;另用脂质体转染构建APP695高表达CHO细胞系,用G418(500mg/L)对转染后细胞进行筛选,最终获得表达人CHO-APP695细胞模型,Western-blot法检测APP-N端片段蛋白的表达。用0.1μmol/L的硒、锌处理转染细胞,48h后,ELISA法检测细胞培养上清中Aβ1-40含量,Western-blot法检测细胞裂解液APP-N端片段蛋白的表达。结果 Western-blot法检测显示CHO-APP695细胞高表达APP-N端片段蛋白;与对照组比较,硒、锌处理组抑制内源性γ-内切酶的活性,且硒、锌联合使用,抑制作用增强;Aβ1-40的含量也减少;单纯硒、锌和硒锌联合处理组,APP-N端片段蛋白表达水平增加。结论硒、锌抑制内源性γ-内切酶对APP蛋白的剪切,减少Aβ1-40的生成,可能对阿尔茨海默病的发病及进展有一定的预防保护作用。

Objective To study the effects of selenium and zinc on amyloid precursor protein （APP） in senile dementia model cell. Method 0.1μmol/L selenium and 0.1μmol/L zinc were added to primary cultured hippocampus neuron cells isolated from fetal rats. The activity of Y-secretase was measured by enzyme-linked immunosorbent assay （ELISA）. In addition, the APP695 gene was transfected into CHO cells and stably transfected cell clones were screened by G418 （500mg/L）.After treatment with selenium and zinc for 48h, the APP-N terminal fragment protein expression was identified by Western-blot analysis and Aβ 1-40 concentration was detected by ELISA in the supernatants of cell culture. Results Western-blot assay showed highly expression of APP-N terminal fragment protein in CHO-APP695 ceils, indicating that the APP695-overexpression CHO cell line was established. Compared with control group, the results of ELISA analysis showed that selenium and zinc could inhibit the activity of ~ -secretase, and the combination of selenium and zinc could decrease the concentration of A β 1-40 more significantly. Selenium, zinc combination of selenium and zinc increased the APP-N terminal fragment protein expression. Conclusion Selenium and zinc have potential protection against Alzheimer＇s disease by inhibiting the activity of y-secretase and decreasing A β 1-40 production.