本文選題：姜黃素 + 淀粉樣蛋白前體　； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：【研究背景和目的】阿爾茨海默氏病(Alzheimer’s disease,AD)是最常見(jiàn)的老年癡呆類型。AD發(fā)病的病理核心是老年斑中β樣淀粉樣蛋白(β-amyloid,Aβ)的沉積,神經(jīng)元纖維纏結(jié)(NFTs),大腦皮層和海馬區(qū)突觸聯(lián)系缺失等。Aβ由淀粉樣蛋白前體(Amyloid precursor protein,APP)經(jīng)β-分泌酶(BACE 1)和γ-分泌酶酶切后生成。Aβ易聚集形成寡聚體、纖維化、沉積為粥樣斑塊,對(duì)神經(jīng)元造成損害,因此抑制Aβ的生成是治療AD的關(guān)鍵。目前臨床上AD的治療主要是膽堿酯酶抑制劑、NMDA受體拮抗劑等改善癥狀的藥物,尚無(wú)特異性針對(duì)病因、預(yù)防和阻滯病情進(jìn)展的藥物。姜黃素是一種從姜科植物姜黃等的根莖中提取的黃色色素,具有抗淀粉樣變、抗炎、抗氧化及抑制膽堿酯酶活性等作用。體外及體內(nèi)的實(shí)驗(yàn)證實(shí)姜黃素可以顯著降低Aβ的量,抑制Aβ的聚集、纖維化和斑塊形成。然而目前AD的研究中使用的大多是姜黃素復(fù)合物,其包含姜黃素(Cur)、去甲氧基姜黃素(DMC)和雙去甲氧基姜黃素(BDMC)三種單體,并且有研究提出姜黃素單體Cur抑制Aβ生成的作用較姜黃素復(fù)合物及余單體明顯,但姜黃素單體Cur抑制Aβ的機(jī)制尚不清楚。微小核糖核酸(micro RNAs,mi RNAs)是一類非編碼的內(nèi)源性小分子RNA,通過(guò)和靶基因m RNA堿基配對(duì)阻礙其翻譯及降解。據(jù)報(bào)道m(xù)i RNAs可參與神經(jīng)變性疾病的病理過(guò)程,能靶向于AD相關(guān)基因的3’非翻譯區(qū)(3’UTR)使其翻譯產(chǎn)物的表達(dá)受抑制。然而在AD研究中尚無(wú)姜黃素單體通過(guò)調(diào)控APP基因特異的mi RNAs而影響其靶基因產(chǎn)物的表達(dá)的報(bào)道。因此,本課題的研究目的是:研究姜黃素單體對(duì)sw APP HEK293細(xì)胞Aβ生成的影響及可能的初步機(jī)制,為姜黃素單體的進(jìn)一步應(yīng)用提供實(shí)驗(yàn)依據(jù)。【實(shí)驗(yàn)方法】設(shè)定姜黃素單體Cur的濃度梯度(2、5、10、20、40μM)作用于sw APP HEK293細(xì)胞各時(shí)間點(diǎn)(6、12、24h)后,采用MTT法(MTT比色法)檢測(cè)細(xì)胞存活及生長(zhǎng),ELISA法(酶聯(lián)免疫吸附試驗(yàn))檢測(cè)Aβ40和Aβ42的水平,篩選出姜黃素的最佳作用濃度及時(shí)間點(diǎn);用RT-PCR(聚合酶鏈?zhǔn)椒磻?yīng))檢測(cè)APPm RNA的表達(dá),Western blot法(聚丙烯酰胺凝膠電泳)檢測(cè)APP蛋白的表達(dá),q RT-PCR(實(shí)時(shí)熒光定量多聚酶鏈反應(yīng))檢測(cè)mi RNA的表達(dá)�！狙芯拷Y(jié)果】1、與對(duì)照組相比,濃度≤5μM的Cur對(duì)細(xì)胞活性無(wú)明顯影響,而濃度≥10μM的姜黃素對(duì)細(xì)胞有毒性作用;2、濃度為5μM的Cur作用細(xì)胞24h后明顯抑制細(xì)胞內(nèi)Aβ40和Aβ42的生成,較各對(duì)照組差異有統(tǒng)計(jì)學(xué)意義;3、Cur對(duì)APP m RNA的表達(dá)無(wú)明顯影響,但明顯抑制APP蛋白的表達(dá),與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義;4、Cur使mi R-153的表達(dá)上調(diào),使mi R-101的表達(dá)下調(diào),與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義,而對(duì)mi R-195的表達(dá)無(wú)明顯影響。5、mi R-153對(duì)APP m RNA的表達(dá)無(wú)明顯影響,可在轉(zhuǎn)錄后水平負(fù)性調(diào)控APP蛋白的表達(dá),與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義。【結(jié)論】1、Cur的濃度及時(shí)間為5μM、24h時(shí),對(duì)sw APP HEK293細(xì)胞中Aβ40和Aβ42生成的抑制作用最明顯;2、Cur可在轉(zhuǎn)錄后水平抑制APP蛋白的表達(dá)從而抑制Aβ40及Aβ42的生成;3、Cur可能通過(guò)誘導(dǎo)mi R-153過(guò)表達(dá)在轉(zhuǎn)錄后水平抑制APP蛋白的表達(dá),從而抑制Aβ40、Aβ42的生成。
[Abstract]:[background and purpose] Alzheimer 's disease (AD) is the most common pathological type of Alzheimer's disease, the core of the pathology is the deposition of beta amyloid (beta -amyloid, A beta) in the senile plaques, neuronal tangles (NFTs), and the absence of synaptic connections in the cerebral cortex and hippocampus, etc.,.A beta precursor (Amylo) (Amylo). ID precursor protein, APP) formation of.A beta after beta secretase (BACE 1) and gamma secretase is easily aggregated to form oligomers, fibrosis, and deposition of atherosclerotic plaques, causing damage to neurons. Therefore, inhibition of the formation of A beta is the key to the treatment of AD. At present, the main clinical AD treatment is the improvement of cholinesterase inhibitor, NMDA receptor antagonist and so on. Curcumin is a yellow pigment extracted from rhizomes of turmeric, such as turmeric, which has anti amyloid, anti-inflammatory, antioxidation and inhibition of cholinesterase activity. In vitro and in vivo experiments confirm that curcumin can significantly reduce the amount of A beta and inhibit the activity of curcumin in vitro and in vivo. The aggregation, fibrosis and plaque formation of A beta are made. However, most of the use of curcumin complexes in AD studies, including curcumin (Cur), normethoxy curcumin (DMC) and dimethoxy curcumin (BDMC) three monomers, has been studied and suggested that the effect of curcumin Cur on the inhibition of A beta generation is more than curcumin complex and residual monopalicine However, the mechanism of curcumin monomer Cur inhibition of A beta is not clear. Micro RNAs (MI RNAs) is a class of non coding endogenous small molecule RNA, which prevents its translation and degradation by pairing the target gene m RNA base. It is reported that MI RNAs can be involved in the pathological process of neurodegenerative diseases and can be targeted to 3 'non translation of AD related genes. The region (3 'UTR) inhibits the expression of its translation products. However, there is no report on the effect of curcumin monomer on the expression of the target gene products by regulating the specific mi RNAs of the APP gene in the study of AD. Therefore, the purpose of this study is to study the effect of curcumin monomer on the A beta formation of SW APP HEK293 cells and the possible preliminary mechanism. The further application of curcumin monomer provides experimental basis. [experimental method] setting the concentration gradient of curcumin monomer Cur (2,5,10,20,40 mu M) acting on SW APP HEK293 cells at every time point (6,12,24h), using MTT (MTT colorimetric method) to detect cell survival and growth, ELISA assay (enzyme-linked immunosorbent assay) for the detection of A beta 40 and A beta 42 levels, The optimum concentration and time point of curcumin were screened, the expression of APPm RNA was detected by RT-PCR (polymerase chain reaction), the expression of APP protein was detected by Western blot (polyacrylamide gel electrophoresis), and the expression of MI RNA was detected by Q RT-PCR (real-time fluorescent quantitative polymerase chain reaction). [results] 1, the concentration was less than 5 mu M compared with the control group. Cur had no obvious effect on cell activity, and the curcumin with a concentration of more than 10 mu M had toxic effects on the cells. 2, 24h of Cur acting cells with a concentration of 5 mu M obviously inhibited the formation of A beta 40 and A beta 42 in the cells, compared with the control group, 3, Cur had no obvious influence on the expression of APP m RNA, but obviously inhibited the expression of APP protein and compared with the control. 4, Cur increased the expression of MI R-153 and reduced the expression of MI R-101, compared with the control group, but there was no significant difference in the expression of MI R-195. Mi R-153 had no obvious influence on the expression of APP m, but the expression of the protein in the posttranscriptional water level was different from that of the control group. [Conclusion] [Conclusion] 1, the concentration and time of Cur are 5 mu M, 24h, the inhibition of A beta 40 and A beta 42 in SW APP HEK293 cells is most obvious; 2, Cur can inhibit the expression of APP protein at the post transcriptional level and inhibit the production of A beta 40 and A beta 42. 3 It inhibits the formation of A beta 40, A beta 42.
【學(xué)位授予單位】：廣州醫(yī)科大學(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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