细胞内pH改变与β分泌酶关系的研究
发布时间:2018-04-03 18:33
本文选题:阿尔茨海默病 切入点:β-分泌酶 出处:《辽宁医学院》2012年硕士论文
【摘要】:目的 本文旨在研究当细胞内环境呈酸化改变时,,细胞内β-分泌酶mRNA表达、蛋白含量及活性的变化,进而说明细胞内pH值变化与β-分泌酶的关系。 方法 培养人神经母细胞瘤细胞(Human neuroblastoma cell line, SH-SY5Y),将构建好的靶向人NHE-1基因的siRNA腺病毒及腺病毒空载体感染SH-SY5Y细胞,建立NHE-1基因敲低细胞模型及负对照组;用配制好的无血清的培养基培养SH-SY5Y细胞,收集前5天培养的细胞,建立无血清培养组。实验分成4组,即正常对照组、NHE-1基因敲低组、负对照组和无血清培养组。运用荧光分光光度计测定各组细胞内pH值,采用Real-time PCR技术检测各组细胞内β-分泌酶mRNA表达,通过Western Blotting检测β-分泌酶蛋白表达水平,通过ELISA检测β-分泌酶的活性变化。利用单因素方差分析及LSD-t检验统计分析各组细胞的检测指标差异情况。 结果 无血清条件下培养细胞,当培养4天时细胞状态最佳。荧光探针法测得NHE-1基因敲低组细胞内pH值为6.91±0.055,无血清培养组细胞内pH值为7.03±0.062,负对照组胞内pH值为7.22±0.026,正常对照组细胞内pH值为7.19±0.066,与正常对照组和负对照组相比,NHE-1基因敲低组和无血清培养组细胞内pH值明显降低,差异具有统计学意义(p0.05),而正常对照组和负对照组相比细胞内pH值无明显变化,差异无统计学意义(p0.05)。Real-time PCR及Western Blotting结果显示:NHE-1基因敲低组细胞内β-分泌酶mRNA表达和蛋白含量分别为5.65±0.33和0.37±0.030,无血清培养组细胞内β-分泌酶mRNA表达和蛋白含量分别为2.40±0.55和0.32±0.031,与正常对照组(0.92±0.34;0.12±0.020)和负对照组(1.00±0.00;0.14±0.021)相比,NHE-1基因敲低组和无血清培养组细胞内β-分泌酶mRNA表达和蛋白含量明显增多,差异具有统计学意义(p0.05),而正常对照组和负对照组相比细胞内β-分泌酶mRNA表达和蛋白含量无明显变化,差异无统计学意义(p0.05)。ELISA结果显示:NHE-1基因敲低组细胞内β-分泌酶的蛋白水平为449.45±65.95ng/L,无血清培养组细胞内β-分泌酶的蛋白水平为468.60±58.59ng/L,与正常对照组(290.65±82.33ng/L)和负对照组(327.11±82.32ng/L)相比,NHE-1基因敲低组和无血清培养组细胞内β-分泌酶的活性明显增强,差异具有统计学意义(p0.05),而正常对照组和负对照组相比细胞内β-分泌酶的活性无明显变化,差异无统计学意义(p0.05)。 结论 通过成功构建NHE-1基因敲低组及无血清培养组,使细胞内环境呈酸化改变,证实细胞内pH降低能够使β-分泌酶的表达上调并增强其活性,进而可能影响APP水解剪切,促进Aβ的生成积聚。
[Abstract]:objective
The aim of this study is to investigate the expression of mRNA, the protein content and activity of beta secretase in cells when the intracellular environment is acidified, and to further illustrate the relationship between pH value and beta secretase.
Method
Cultured human neuroblastoma cells (Human neuroblastoma cell line, SH-SY5Y siRNA), adenovirus and adenovirus constructed targeting human NHE-1 gene in SH-SY5Y cells infected with NHE-1 gene knockdown cell model is established and the negative control group; SH-SY5Y cells cultured with the prepared serum-free culture medium 5 days before the collection of cells, establish the serum-free group. The experiment was divided into 4 groups: normal control group, NHE-1 knockdown group, negative control group and serum-free group. Spectrophotometric determination of the intracellular pH value by fluorescence, using Real-time PCR technology to detect the intracellular beta secretase mRNA the expression level of Western Blotting expression by detecting beta secretase protein, the activity of ELISA in the detection of beta secretase. Using single factor analysis of variance and LSD-t test statistical analysis indexes of cells were difference.
Result
Cells cultured in serum-free conditions, when cells cultured for 4 days. The best knockdown group of intracellular pH 6.91 + 0.055 NHE-1 gene measured by fluorescence probe, no group of intracellular pH 7.03 + 0.062 serum culture negative group, the intracellular pH value was 7.22 + 0.026 group, normal control group cells the pH value is 7.19 + 0.066, compared with the normal control group and negative control group, NHE-1 knockdown group and serum-free group of intracellular pH levels decreased significantly, the difference was statistically significant (P0.05), and the normal control group the intracellular pH values had no significant changes compared with the negative control group, the difference was not statistically significant (P0.05).Real-time PCR and Western Blotting showed that NHE-1 mutant enzyme mRNA expression and protein content were low in beta cell secretion group were 5.65 + 0.33 and 0.37 + 0.030, serum-free group of intracellular beta secretase mRNA expression and protein content were 2.40 + 0.55 and 0.32 + 0 .031, and the normal control group (0.92 + 0.34; 0.12 + 0.020) and negative control group (1 + 0; 0.14 + 0.021) compared with NHE-1 gene knockdown group and serum-free group of intracellular beta secretase mRNA expression and protein content increased, the difference was statistically significant (P0.05), and normal the control group of intracellular beta secretase mRNA expression and protein content had no significant change compared with the negative control group, the difference was not statistically significant (P0.05).ELISA showed that NHE-1 mutant protein level low intracellular beta secretase is 449.45 + 65.95ng/L, no intracellular beta secretase serum protein level the 468.60 + 58.59ng/L, and normal control group (290.65 + 82.33ng/L) and negative control group (327.11 + 82.32ng/L) compared with NHE-1 gene knockdown group and serum-free group of intracellular beta secretase activity increased significantly, the difference was statistically significant (P0.05), and normal control There was no significant difference in the activity of intracellular beta secretase in the group and the negative control group, and there was no significant difference between the two groups (P0.05).
conclusion
Through successful construction of NHE-1 knockdown group and serum-free culture group, the intracellular environment is acidified. It is confirmed that the decrease of intracellular pH can increase the expression of beta secretase and enhance its activity, which may affect APP hydrolysis and shear, and promote the accumulation of A beta.
【学位授予单位】:辽宁医学院
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R749.16
【参考文献】
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