U18666A对星形胶质瘤U373细胞APP代谢及Aβ生成的影响(1)
[摘要] 目的 观察胆固醇转运抑制剂U18666A(UA)对星形胶质瘤U373细胞淀粉样前体蛋白(APP)代谢及β淀粉样蛋白(Aβ)生成的影响。 方法 0、1、3、5 μg/mL UA 作用U373细胞24 h，或5 μg/mL UA与U373细胞共培养0、12、24、48 h，Western blot检测APP及其代谢产物α-CTF、β-CTF的表达，酶联免疫吸附试验检测细胞内Aβ1-40含量，荧光酶法检测β-分泌酶活性。 结果 3、5 μg/mL UA组APP蛋白表达水平、β-CTF和α-CTF蛋白表达量、Aβ1-40含量高于0 μg/mL UA组，差异均有统计学意义(P < 0.05或P < 0.01)。共培养24、48 h组APP表达水平、β-CTF和α-CTF蛋白表达量、Aβ1-40含量高于共培养0 h组，差异均有高度统计学意义(均P < 0.01)。5 μg/mL UA组β-分泌酶活性高于对照组，差异有高度统计学意义(P < 0.01)。 结论 UA通过增加β-分泌酶活性增强APP的表达和代谢，促进Aβ1-40的生成，加速阿尔茨海默病进程。
[关键词] 胆固醇;U18666A;U373细胞;淀粉样前体蛋白;β淀粉样蛋白
[中图分类号] R749.16 [文献标识码] A [文章编号] 1673-7210(2020)08(a)-0004-04
[Abstract] Objective To investigate the effect of cholesterol transport inhibitor U18666A (UA) on amyloid precursor protein (APP) metabolism and β-amyloid protein (Aβ) generation in U373 cells of astroglioma. Methods U373 cells were treated with UA at the dose of zero， one， three and five μg/mL for 24 h or 5 μg/mL UA for different times (0， 12， 24， 48 h). The expressions of APP and its metabolites α-CTF and β-CTF were detected using Western blot. The cellular levels of Aβ1-40 were measured with enzyme linked immunosorbent assay and the activity of β-secretase was determined by fluorescence assay. Results The expression levels of APP， α-CTF and β-CTF， and Aβ1-40 in 5 μg/mL UA group were higher than those in 0 μg/mL UA group， and the differences were statistically significant (P < 0.05 or P < 0.01). The expression levels of APP， α-CTF and β-CTF， and Aβ1-40 in co-cultured 24 h and 48 h groups were significantly higher than those in co-cultured 0 h group， and the differences were highly statistically significant (all P < 0.01). The activity of β-secretase in 5 μg/mL UA group was significantly higher than that in the control group (P < 0.01). Conclusion UA enhances APP expression and metabolism by increasing β-secretase activity， promotes Aβ1-40 generation， accelerates the pathological process of Alzheimer′s disease.
[Key words] Cholesterol; U18666A; U373 cell; Amyloid precursor protein; β-amyloid protein
阿爾茨海默病(Alzheimer′s disease，AD)是一种常见的中枢神经系统退行性疾病，患者脑内有明显的β淀粉样蛋白(Aβ)沉积。Aβ主要由淀粉样前体蛋白(APP)经β-分泌酶和γ-分泌酶水解产生[1-2]。胆固醇水平偏高患者AD发病风险明显升高[3-6]，胆固醇代谢与AD关系是研究AD发病机制的一个热点。本研究以星形胶质瘤U373细胞为研究对象，加入不同剂量胆固醇转运抑制剂U18666A(UA)作用不同时间，观察UA对U373细胞APP代谢及Aβ生成的影响，为进一步理解胆固醇代谢与AD关系提供实验依据。
1 材料与方法
1.1 仪器与试剂
Thermo 3001酶标仪(Thermo);Power Poc Basic电泳仪(Bio-Rad);ChemiDoc MP全功能荧光化学发光成像仪(Bio-Rad);UA[爱必信(上海)生物科技有限公司，MZ28];Y188抗体(Abcam，ab32136);Aβ1-40试剂盒(Life Technologies，KHB3482);β-分泌酶活性荧光检测试剂盒(Abcam，ab65357);β-actin抗体(ZM-0001)、辣根酶标记山羊抗兔IgG(ZDR-5403)、辣根酶标记山羊抗小鼠IgG(ZB-2305)均购自北京中杉金桥生物有限公司。, http://www.100md.com(杨宏艳 都晓辉 包亚男)
[关键词] 胆固醇;U18666A;U373细胞;淀粉样前体蛋白;β淀粉样蛋白
[中图分类号] R749.16 [文献标识码] A [文章编号] 1673-7210(2020)08(a)-0004-04
[Abstract] Objective To investigate the effect of cholesterol transport inhibitor U18666A (UA) on amyloid precursor protein (APP) metabolism and β-amyloid protein (Aβ) generation in U373 cells of astroglioma. Methods U373 cells were treated with UA at the dose of zero， one， three and five μg/mL for 24 h or 5 μg/mL UA for different times (0， 12， 24， 48 h). The expressions of APP and its metabolites α-CTF and β-CTF were detected using Western blot. The cellular levels of Aβ1-40 were measured with enzyme linked immunosorbent assay and the activity of β-secretase was determined by fluorescence assay. Results The expression levels of APP， α-CTF and β-CTF， and Aβ1-40 in 5 μg/mL UA group were higher than those in 0 μg/mL UA group， and the differences were statistically significant (P < 0.05 or P < 0.01). The expression levels of APP， α-CTF and β-CTF， and Aβ1-40 in co-cultured 24 h and 48 h groups were significantly higher than those in co-cultured 0 h group， and the differences were highly statistically significant (all P < 0.01). The activity of β-secretase in 5 μg/mL UA group was significantly higher than that in the control group (P < 0.01). Conclusion UA enhances APP expression and metabolism by increasing β-secretase activity， promotes Aβ1-40 generation， accelerates the pathological process of Alzheimer′s disease.
[Key words] Cholesterol; U18666A; U373 cell; Amyloid precursor protein; β-amyloid protein
阿爾茨海默病(Alzheimer′s disease，AD)是一种常见的中枢神经系统退行性疾病，患者脑内有明显的β淀粉样蛋白(Aβ)沉积。Aβ主要由淀粉样前体蛋白(APP)经β-分泌酶和γ-分泌酶水解产生[1-2]。胆固醇水平偏高患者AD发病风险明显升高[3-6]，胆固醇代谢与AD关系是研究AD发病机制的一个热点。本研究以星形胶质瘤U373细胞为研究对象，加入不同剂量胆固醇转运抑制剂U18666A(UA)作用不同时间，观察UA对U373细胞APP代谢及Aβ生成的影响，为进一步理解胆固醇代谢与AD关系提供实验依据。
1 材料与方法
1.1 仪器与试剂
Thermo 3001酶标仪(Thermo);Power Poc Basic电泳仪(Bio-Rad);ChemiDoc MP全功能荧光化学发光成像仪(Bio-Rad);UA[爱必信(上海)生物科技有限公司，MZ28];Y188抗体(Abcam，ab32136);Aβ1-40试剂盒(Life Technologies，KHB3482);β-分泌酶活性荧光检测试剂盒(Abcam，ab65357);β-actin抗体(ZM-0001)、辣根酶标记山羊抗兔IgG(ZDR-5403)、辣根酶标记山羊抗小鼠IgG(ZB-2305)均购自北京中杉金桥生物有限公司。, http://www.100md.com(杨宏艳 都晓辉 包亚男)