【摘要】 目的筛选具有提高神经内肽酶(NEP)活性从而降低神经细胞β-淀粉样肽(Aβ)水平的药物,为临床治疗阿尔茨海默症(AD)提供实验依据。方法以脂质体LipofectamineTM2000介导瑞典突变型淀粉样肽前体蛋白(sweAPP)表达质粒pcDNA3.1-sweAPP转染神经母细胞瘤细胞SK-N-SH,G418筛选获得稳定表达细胞株作为AD模型细胞,采用蛋白印迹技术检测sweAPP表达情况,酶联免疫吸附试验(ELISA)检测细胞培养基中A4β0和A4β2含量变化,进而以MTT法检测各天然药物对SK-N-SH细胞增殖能力的影响,确定药物的有效浓度及最佳作用时间,以NEP肽酶实验检测药物对NEP酶活性影响,ELISA法检测药物对Aβ水平的影响。结果Western Blot结果显示,转染后SK-N-SH细胞高表达sweAPP蛋白;ELISA结果显示,转染后的SK-N-SH细胞分泌A4β0、A4β2明显升高,分别为转染前的7.26和3.27倍(P<0.05)。NEP肽酶实验检测结果显示,茶多酚、人参皂苷Rb1、Rg3能够明显提高NEP酶活性,分别为(117±2.8)%(、131±4.0)%和(121±1.6)%。ELISA结果显示,经茶多酚、人参皂苷Rb1、Rg3作用后,细胞培养基中A4β0和Aβ42水平明显降低,分别为对照组的(82±0.78)%和(85±2.21)%(、74±1.61)%和(99±3.31)%、(83±5.31)%和(76±6.74)%。结论成功构建AD模型细胞;茶多酚、人参皂苷Rb1、Rg3可提高NEP酶活性,从而有效降低Aβ的水平;本研究为茶多酚、人参皂苷Rb1、Rg3在临床上的应用以及分子机制研究提供了试验数据。更多还原

【Abstract】 Objective To obtain potential chemicals useful for chemotherapy of Alzheimer′s disease(AD) by screening naturally occurring compounds which could increase the Neprilysin(NEP) activity.Methods Neuroblastoma cells were cultured(SK-N-SH cells) and pcDNA3.1-sweAPP was transfected into the SK-N-SH cells with LipofectamineTM2000,and G418 was used for screening.The stably transfected cells were confirmed with Western blot and enzyme linked immunosobent assay(ELISA). MTT was used to identify the optimal drug concentration and time.The effects of drugs on NEP activity were determined by the NEP peptidase assay,and the Aβ levels were determined by ELISA.Results Western blot results showed that SK-N-SH cells transfected with pcDNA3.1-sweAPP highly expressed APP proteins,and ELISA results showed that Aβ40 and Aβ42 in culture media were significantly increased respectively by 7.26 and 3.27 fold after transfection.NEP peptidase assay showed that tea polyphenols,ginsenoside Rb1 and Rg3 could significantly enhance the NEP activity by 117±2.8%,131±4.0% and 121±1.6% respectively.ELISA results showed that they could significantly decrease Aβ40 and Aβ42 in culture media of SK-N-SH cells by 82±0.78% and 85±2.21%,74±1.61% and 99±3.31% and 83±5.31% and 76±6.74% respectively.Conclusion The AD model cells were successfully constructed,and tea polyphenols,Ginsenoside Rb1 and Rg3 can effectively decrease Aβ40 and Aβ42 in culture media.Our findings offer potent theoretical supports for application of these drugs in treating AD.更多还原