Aβ25-35对C9ORF72基因及蛋白表达影响的体外研究

发布时间：2018-05-13 11:00

本文选题：Aβ25-35 + C9ORF72　；参考：《郑州大学》2017年硕士论文

【摘要】：背景与目的阿尔茨海默病是老年期最常见的痴呆类型,作为发病率最高的神经退行性疾病,随着人口老龄化,其已变成现代社会严重的医学问题。淀粉样蛋白沉积老年斑(SPs)和神经元纤维缠结(NFTs)是AD两个特征性病理改变,且β-淀粉样蛋白是老年斑的主要成分,其产生的神经毒性作用已经被公认为是AD形成和发展的关键因素。C9ORF72(chromosome 9 open reading frame,C9ORF72)基因位于9号染色体,编码12个外显子,但其所编码蛋白的功能尚不十分明确;其非编码区GGGGCC六核苷酸重复扩增已被证实存在于在神经系统退行性疾病中,更是导致肌萎缩侧索硬化和额颞叶痴呆的主要原因。进一步的研究表明C9ORF72上的六碱基重复序列可能在阿尔茨海默病也发挥着致病作用。本研究旨在探讨β-淀粉样肽25-35(Aβ25-35)干预PC12细胞后其对C9ORF72基因和蛋白表达的影响。材料和方法我们采用不同浓度Aβ25-35(0μM、10μM、20μM及40μM)处理PC12细胞48小时后,采用AnnexinV/PI检测细胞凋亡情况;实时定量PCR法(Quantative Real-time PCR,qRT-PCR)检测经不同浓度Aβ25-35处理后PC12细胞中C9ORF72基因全长的表达情况;蛋白免疫印迹法(Western blotting)检测PC12细胞中C9ORF72蛋白的表达。结果(1)PC12细胞凋亡的检测:散点图上,相比于对照组,随着Aβ25-35浓度增加,右上象限凋亡细胞数明显增加且细胞凋亡率显著提高;且10μM、20μM及40μM组相比对照组差异均具有显著性统计学意义(P0.01)。(2)C9ORF72基因的表达:对照组C9ORF72基因2-ΔΔCt值为0.651±0.016,10μM组为0.674±0.123,20μM组为1.000±0.075,40μM组为2.839±0.199,由此可见,随着Aβ25-35浓度的增加,mRNA相对水平有所增加,且20μM及40μM组相比对照组有显著性统计学差异(P0.001)。(3)C9ORF72蛋白的表达:不同浓度Aβ25-35(0μM、10μM、20μM及40μM)处理PC12细胞后均有C9ORF72蛋白表达;对照组C9ORF72蛋白表达量为0.400±0.015,10u M Aβ25-35组蛋白表达量为0.430±0.015,20uM组蛋白表达量为0.520±0.025,40uM组蛋白表达量为0.920±0.053。随着Aβ25-35浓度增加,蛋白表达水平增加,且20uM组及40uM组蛋白表达量均高于对照组,且具有显著性统计学差异(P0.01)。结论(1)Aβ25-35对PC12细胞具有致凋亡作用。(2)Aβ25-35可导致C9ORF72基因及蛋白高表达,可能是阿尔茨海默病的致病机制之一。
[Abstract]:Background & objective Alzheimer's disease is the most common type of dementia in the elderly. As a neurodegenerative disease with the highest incidence, Alzheimer's disease has become a serious medical problem in modern society with the aging of the population. Amyloid deposition (SPs) and neurofibrillary tangles (NFTs) are two characteristic pathological changes of AD, and 尾 -amyloid protein is the main component of senile plaque. Its neurotoxic effect has been recognized as the key factor for the formation and development of AD. The C9ORF72Ly 9 open reading frame C9ORF72) gene is located on chromosome 9 and encodes 12 exons, but the function of the encoded protein is not very clear. The repeated amplification of GGGGCC in the noncoding region has been proved to exist in neurodegenerative diseases and is the main cause of amyotrophic lateral sclerosis and frontotemporal dementia. Further studies suggest that six-base repeats on C9ORF72 may also play a role in Alzheimer's disease. The aim of this study was to investigate the effect of 尾 -amyloid peptide 25-35A 尾 25-35 on the expression of C9ORF72 gene and protein in PC12 cells. Materials and methods PC12 cells were treated with different concentrations of A 尾 25-35 0 渭 M 10 渭 M 20 渭 M and 40 渭 M for 48 hours, and AnnexinV/PI was used to detect the apoptosis of PC12 cells. The expression of C9ORF72 gene in PC12 cells treated with different concentrations of A 尾 25-35 was detected by real-time quantitative PCR method. Western blotting was used to detect the expression of C9ORF72 protein in PC12 cells. Results: compared with the control group, the number of apoptotic cells in the right upper quadrant and the apoptotic rate in the right quadrant increased significantly with the increase of A 尾 25-35 concentration. There was significant difference between 10 渭 M and 40 渭 M groups in the expression of C9ORF72 gene: in the control group, the expression of C9ORF72 gene 2- 螖 Ct was 0.651 卤0.016 渭 M, 0.674 卤0.123 渭 M vs 1.000 卤0.07540 渭 M, 2.839 卤0.199, which showed that the relative level of C9ORF72 mRNA increased with the increase of A 尾 25-35 concentration. There was significant difference between 20 渭 M and 40 渭 M groups in the expression of C9ORF72 protein. The expression of C9ORF72 protein was observed in PC12 cells treated with 10 渭 M 10 渭 M and 40 渭 M of A 尾 25-35 渭 M at different concentrations. The expression of C9ORF72 protein in the control group was 0.400 卤0.015 ~ 10u Ma 尾 25-35, 0.430 卤0.015 ~ 20uM, 0.520 卤0.025 ~ 40uM, 0.920 卤0.053. With the increase of A 尾 25-35 concentration, the level of protein expression increased, and the expression of protein in 20uM group and 40uM group were higher than that in control group, and there was significant statistical difference (P 0.01). Conclusion A 尾 25-35 can induce apoptosis in PC12 cells. The high expression of C9ORF72 gene and protein may be one of the pathogenetic mechanisms of Alzheimer's disease.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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