本文選題：姜黃素 + 淀粉樣蛋白前體��； 參考：《廣州醫(yī)科大學》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,神經元纖維纏結(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,對神經元造成損害,因此抑制Aβ的生成是治療AD的關鍵。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,尚無特異性針對病因、預防和阻滯病情進展的藥物。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。體外及體內的實驗證實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合物及余單體明顯,但姜黃素單體Cur抑制Aβ的機制尚不清楚。微小核糖核酸(micro RNAs,mi RNAs)是一類非編碼的內源性小分子RNA,通過和靶基因m RNA堿基配對阻礙其翻譯及降解。據報道m(xù)i RNAs可參與神經變性疾病的病理過程,能靶向于AD相關基因的3’非翻譯區(qū)(3’UTR)使其翻譯產物的表達受抑制。然而在AD研究中尚無姜黃素單體通過調控APP基因特異的mi RNAs而影響其靶基因產物的表達的報道。因此,本課題的研究目的是:研究姜黃素單體對sw APP HEK293細胞Aβ生成的影響及可能的初步機制,為姜黃素單體的進一步應用提供實驗依據�！緦嶒灧椒ā吭O定姜黃素單體Cur的濃度梯度(2、5、10、20、40μM)作用于sw APP HEK293細胞各時間點(6、12、24h)后,采用MTT法(MTT比色法)檢測細胞存活及生長,ELISA法(酶聯(lián)免疫吸附試驗)檢測Aβ40和Aβ42的水平,篩選出姜黃素的最佳作用濃度及時間點;用RT-PCR(聚合酶鏈式反應)檢測APPm RNA的表達,Western blot法(聚丙烯酰胺凝膠電泳)檢測APP蛋白的表達,q RT-PCR(實時熒光定量多聚酶鏈反應)檢測mi RNA的表達�！狙芯拷Y果】1、與對照組相比,濃度≤5μM的Cur對細胞活性無明顯影響,而濃度≥10μM的姜黃素對細胞有毒性作用;2、濃度為5μM的Cur作用細胞24h后明顯抑制細胞內Aβ40和Aβ42的生成,較各對照組差異有統(tǒng)計學意義;3、Cur對APP m RNA的表達無明顯影響,但明顯抑制APP蛋白的表達,與對照組相比差異有統(tǒng)計學意義;4、Cur使mi R-153的表達上調,使mi R-101的表達下調,與對照組相比差異有統(tǒng)計學意義,而對mi R-195的表達無明顯影響。5、mi R-153對APP m RNA的表達無明顯影響,可在轉錄后水平負性調控APP蛋白的表達,與對照組相比差異有統(tǒng)計學意義。【結論】1、Cur的濃度及時間為5μM、24h時,對sw APP HEK293細胞中Aβ40和Aβ42生成的抑制作用最明顯;2、Cur可在轉錄后水平抑制APP蛋白的表達從而抑制Aβ40及Aβ42的生成;3、Cur可能通過誘導mi R-153過表達在轉錄后水平抑制APP蛋白的表達,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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16

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