姜黄素单体对swAPP HEK293细胞Aβ生成的影响

发布时间：2018-06-13 09:49

本文选题：姜黄素 + 淀粉样蛋白前体　；参考：《广州医科大学》2017年硕士论文

【摘要】：【研究背景和目的】阿尔茨海默氏病(Alzheimer’s disease,AD)是最常见的老年痴呆类型。AD发病的病理核心是老年斑中β样淀粉样蛋白(β-amyloid,Aβ)的沉积,神经元纤维缠结(NFTs),大脑皮层和海马区突触联系缺失等。Aβ由淀粉样蛋白前体(Amyloid precursor protein,APP)经β-分泌酶(BACE 1)和γ-分泌酶酶切后生成。Aβ易聚集形成寡聚体、纤维化、沉积为粥样斑块,对神经元造成损害,因此抑制Aβ的生成是治疗AD的关键。目前临床上AD的治疗主要是胆碱酯酶抑制剂、NMDA受体拮抗剂等改善症状的药物,尚无特异性针对病因、预防和阻滞病情进展的药物。姜黄素是一种从姜科植物姜黄等的根茎中提取的黄色色素,具有抗淀粉样变、抗炎、抗氧化及抑制胆碱酯酶活性等作用。体外及体内的实验证实姜黄素可以显著降低Aβ的量,抑制Aβ的聚集、纤维化和斑块形成。然而目前AD的研究中使用的大多是姜黄素复合物,其包含姜黄素(Cur)、去甲氧基姜黄素(DMC)和双去甲氧基姜黄素(BDMC)三种单体,并且有研究提出姜黄素单体Cur抑制Aβ生成的作用较姜黄素复合物及余单体明显,但姜黄素单体Cur抑制Aβ的机制尚不清楚。微小核糖核酸(micro RNAs,mi RNAs)是一类非编码的内源性小分子RNA,通过和靶基因m RNA碱基配对阻碍其翻译及降解。据报道mi RNAs可参与神经变性疾病的病理过程,能靶向于AD相关基因的3’非翻译区(3’UTR)使其翻译产物的表达受抑制。然而在AD研究中尚无姜黄素单体通过调控APP基因特异的mi RNAs而影响其靶基因产物的表达的报道。因此,本课题的研究目的是:研究姜黄素单体对sw APP HEK293细胞Aβ生成的影响及可能的初步机制,为姜黄素单体的进一步应用提供实验依据。【实验方法】设定姜黄素单体Cur的浓度梯度(2、5、10、20、40μM)作用于sw APP HEK293细胞各时间点(6、12、24h)后,采用MTT法(MTT比色法)检测细胞存活及生长,ELISA法(酶联免疫吸附试验)检测Aβ40和Aβ42的水平,筛选出姜黄素的最佳作用浓度及时间点;用RT-PCR(聚合酶链式反应)检测APPm RNA的表达,Western blot法(聚丙烯酰胺凝胶电泳)检测APP蛋白的表达,q RT-PCR(实时荧光定量多聚酶链反应)检测mi RNA的表达。【研究结果】1、与对照组相比,浓度≤5μM的Cur对细胞活性无明显影响,而浓度≥10μM的姜黄素对细胞有毒性作用;2、浓度为5μM的Cur作用细胞24h后明显抑制细胞内Aβ40和Aβ42的生成,较各对照组差异有统计学意义;3、Cur对APP m RNA的表达无明显影响,但明显抑制APP蛋白的表达,与对照组相比差异有统计学意义;4、Cur使mi R-153的表达上调,使mi R-101的表达下调,与对照组相比差异有统计学意义,而对mi R-195的表达无明显影响。5、mi R-153对APP m RNA的表达无明显影响,可在转录后水平负性调控APP蛋白的表达,与对照组相比差异有统计学意义。【结论】1、Cur的浓度及时间为5μM、24h时,对sw APP HEK293细胞中Aβ40和Aβ42生成的抑制作用最明显;2、Cur可在转录后水平抑制APP蛋白的表达从而抑制Aβ40及Aβ42的生成;3、Cur可能通过诱导mi R-153过表达在转录后水平抑制APP蛋白的表达,从而抑制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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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