NHE-1 miRNA转染所致细胞内pH值变化对BACE1的影响

摘要

Objective To transfect the adenovirus mediated NHE-1 miRNA into human neuroblastoma(SH-SY5Y), and investigate the expression of BACE1 with the condition of the change of intracellular pH value. Methods The human neuroblastoma (SH-SY5Y) cell were cultured, enrolled and randomly divided into 3 groups, named as normal control group, NHE-1 gene knock down group and negative control group, and structured the NHE-1 gene knock down model. The intracellular pH value was detected in each group by fluorescence spectrophotometer, cells were examined for mRNA and protein expression of BACE1 by Real-time PCR and Western blotting. Results Compared with control group, the intracel-lular pH value was significantly lower in NHE-1 gene knock down group by fluorescent probe method (P <0.01) , while it had no change in negative control group; furthermore, the BACE1 mRNA expression and protein level were significantly higher in NHE-1 gene knock down group than those in paired normal control group (P <0.01), the BACE1 mRNA expres-sion and protein level had no change in negative control group. Conclusions The lower intracellular pH value condition is responsible for the higher BACE1 expression.%目的 探讨腺病毒介导的人Na+-H+交换体-1(NHE-1) miRNA转染人源性神经母细胞瘤细胞株(SHSY5Y)后,细胞内pH值变化及其对β位淀粉样前体蛋白裂解酶1(BACE1)的影响.方法 培养SH-SY5Y,随机分为正常对照组、NHE-1基因敲低组和阴性对照组,并构建NHE-1基因敲低细胞模型.采用荧光分光光度计测定各组细胞内pH值,通过Real-time PCR及Western blotting检测BACE1 mRNA及蛋白表达.结果 与正常对照组相比,NHE-1基因敲低组细胞内pH值明显降低(P＜0.01),阴性对照组细胞内pH值无明显变化；NHE-1基因敲低组细胞内BACE1 mRNA表达及蛋白含量明显增高(P＜0.01),阴性对照组细胞内BACE1 mRNA表达及蛋白含量无明显变化.结论 细胞内pH值降低能够增强BACE1的表达.