摘要
目的研究细胞内pH值下降的情况下,β分泌酶和早老素-1的表达及活性的变化。方法培养人源性神经母细胞瘤(SH-SY5Y)细胞,用RNA干扰技术和无血清培养分别制造细胞内酸化模型;实验分成4组,即正常细胞组、NHE-1基因敲低组、阴性对照组、无血清培养组。运用荧光分光光度计测定各组细胞内pH值,并通过Real-Time PCR和Western Blotting分别检测β分泌酶和早老素-1 mRNA及蛋白的相对表达量,通过ELISA检测β分泌酶和早老素-1的活性。结果 NHE-1基因敲低组及无血清培养组较正常细胞内pH值降低(P<0.05),阴性对照组细胞内pH值无明显变化;与正常、阴性对照组相比,NHE-1基因敲低组β分泌酶mRNA表达为5.65±0.33,蛋白表达为0.37±0.030;早老素-1 mRNA表达1.75±0.04,蛋白表达分别为0.70±0.01,无血清培养组β分泌酶mRNA表达为2.40±0.55,蛋白表达为0.32±0.031;早老素-1 mRNA表达1.39±0.03,蛋白表达分别为0.68±0.08,其表达及活性均明显增强(P<0.05);阴性对照组细胞与正常细胞比较,β分泌酶和早老素-1 mRNA和蛋白表达及活性均无明显变化(P>0.05)。结论细胞内pH降低能够增强β分泌酶的表达和活性;因早老素-1被认为是γ-分泌酶的主要成分和活性中心,因此推测细胞内pH降低也能够增强γ-分泌酶的表达和活性,从而影响APP的剪切,增加Aβ生成。
Objective To investigate the expression and activity changes of β-secretase and presenilin-1 on the condition of intracellular pH decreased. Methods The human neuroblastoma（SH-SYSY）ceils were cultured. The intracellu- lar acidification cell models were established by transfecting recombinant adenovirus interfering plasmid and culturing with the serum-free culture medium. There were four groups of ceils including normal control group, NHE-1 gene knock-down group, negative control group and serum-free cultured group. The intracellular pH values were detected by fluorescence speetrophotometer in each group. The protein and mRNA expression of β-secretase and presenilin-1 were detected by West- ern blotting and Real-Time PCR respectively. Their activities were examined by ELISA. Results Compared with normal control group, the intracellular pH value were significantly decreased in the NHE-1 gene knock-down group and serum-flee cultured group by fluorescent probe method（ P 〈0.05 ） ,while it had no change in negative control group（ P 〉 0.05 ）. The expression of β-secretase mRNA and protein in the NHE-1 gene knock-down group was 5.65 ＋0.33 and 0.37 ＋0. 030 respectively. The expression of Presenilin-1 mRNA and protein in the NHE-1 gene knock-down group was 1.75 ± 0.04 and 0.70 ＋0.01. The expression of β-secretase mRNA and protein in the serum-free cultured group was 2.40 ±0.55 and 0.32 ± 0.031. The expression of Presenilin-1 mRNA and protein in the serum-free cultured group was 1.39 ± 0.03 and 0.68 ＋ 0.08. We found that the mRNA and protein level and activity of β-secretase and Presenilin-1 were significantly higher in the NHE-1 gene knock-down group and serum-free cultured group than that in normal control group（P 〈 0.05）. Furthermore, compared with normal control group, the mRNA and protein level and activity of β-secretase and Presenilin-I had no change in negative control group（ P 〉 0.05 ）. Conclusion Decreased intracellular pH condition contributes to the increased β- secretase expression and activity. Presenilin-1 was considered to be the major component and activity center of-secretase at present, which suggests that decreased intracellular pH could stimulate the activity and expression of-secretase. All these changes may produce more Aβ as a result.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2012年第5期388-392,共5页
Journal of Apoplexy and Nervous Diseases
基金
国家自然科学基金项目(No.81100962)
