本文選題：Aβ25-35 + C9ORF72��； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景與目的阿爾茨海默病是老年期最常見的癡呆類型,作為發(fā)病率最高的神經(jīng)退行性疾病,隨著人口老齡化,其已變成現(xiàn)代社會(huì)嚴(yán)重的醫(yī)學(xué)問題。淀粉樣蛋白沉積老年斑(SPs)和神經(jīng)元纖維纏結(jié)(NFTs)是AD兩個(gè)特征性病理改變,且β-淀粉樣蛋白是老年斑的主要成分,其產(chǎn)生的神經(jīng)毒性作用已經(jīng)被公認(rèn)為是AD形成和發(fā)展的關(guān)鍵因素。C9ORF72(chromosome 9 open reading frame,C9ORF72)基因位于9號(hào)染色體,編碼12個(gè)外顯子,但其所編碼蛋白的功能尚不十分明確;其非編碼區(qū)GGGGCC六核苷酸重復(fù)擴(kuò)增已被證實(shí)存在于在神經(jīng)系統(tǒng)退行性疾病中,更是導(dǎo)致肌萎縮側(cè)索硬化和額顳葉癡呆的主要原因。進(jìn)一步的研究表明C9ORF72上的六堿基重復(fù)序列可能在阿爾茨海默病也發(fā)揮著致病作用。本研究旨在探討β-淀粉樣肽25-35(Aβ25-35)干預(yù)PC12細(xì)胞后其對(duì)C9ORF72基因和蛋白表達(dá)的影響。材料和方法我們采用不同濃度Aβ25-35(0μM、10μM、20μM及40μM)處理PC12細(xì)胞48小時(shí)后,采用AnnexinV/PI檢測細(xì)胞凋亡情況;實(shí)時(shí)定量PCR法(Quantative Real-time PCR,qRT-PCR)檢測經(jīng)不同濃度Aβ25-35處理后PC12細(xì)胞中C9ORF72基因全長的表達(dá)情況;蛋白免疫印跡法(Western blotting)檢測PC12細(xì)胞中C9ORF72蛋白的表達(dá)。結(jié)果(1)PC12細(xì)胞凋亡的檢測:散點(diǎn)圖上,相比于對(duì)照組,隨著Aβ25-35濃度增加,右上象限凋亡細(xì)胞數(shù)明顯增加且細(xì)胞凋亡率顯著提高;且10μM、20μM及40μM組相比對(duì)照組差異均具有顯著性統(tǒng)計(jì)學(xué)意義(P0.01)。(2)C9ORF72基因的表達(dá):對(duì)照組C9ORF72基因2-ΔΔCt值為0.651±0.016,10μM組為0.674±0.123,20μM組為1.000±0.075,40μM組為2.839±0.199,由此可見,隨著Aβ25-35濃度的增加,mRNA相對(duì)水平有所增加,且20μM及40μM組相比對(duì)照組有顯著性統(tǒng)計(jì)學(xué)差異(P0.001)。(3)C9ORF72蛋白的表達(dá):不同濃度Aβ25-35(0μM、10μM、20μM及40μM)處理PC12細(xì)胞后均有C9ORF72蛋白表達(dá);對(duì)照組C9ORF72蛋白表達(dá)量為0.400±0.015,10u M Aβ25-35組蛋白表達(dá)量為0.430±0.015,20uM組蛋白表達(dá)量為0.520±0.025,40uM組蛋白表達(dá)量為0.920±0.053。隨著Aβ25-35濃度增加,蛋白表達(dá)水平增加,且20uM組及40uM組蛋白表達(dá)量均高于對(duì)照組,且具有顯著性統(tǒng)計(jì)學(xué)差異(P0.01)。結(jié)論(1)Aβ25-35對(duì)PC12細(xì)胞具有致凋亡作用。(2)Aβ25-35可導(dǎo)致C9ORF72基因及蛋白高表達(dá),可能是阿爾茨海默病的致病機(jī)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 鹵0.016 渭 M, 0.674 鹵0.123 渭 M vs 1.000 鹵0.07540 渭 M, 2.839 鹵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 鹵0.015 ~ 10u Ma 尾 25-35, 0.430 鹵0.015 ~ 20uM, 0.520 鹵0.025 ~ 40uM, 0.920 鹵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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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