MTERF4对淀粉样前体蛋白代谢途径的影响与机制研究

发布时间：2018-06-17 00:20

本文选题：阿尔茨海默病 + 淀粉样前体蛋白　；参考：《重庆医科大学》2017年博士论文

【摘要】：背景:阿尔茨海默病(Alzheimer’s disease,AD)是一种以进行性的认知障碍、记忆力减退和性格行为改变等症状为主的影响老年人的中枢神经系统退行性疾病。AD由于严重影响患者的生活能力,重症患者甚至日常生活不能自理,给患者家庭和整个社会带来沉重的经济和社会负担。AD的典型特征是在脑中沉积的β-淀粉样蛋白(β-Amyloid peptide,Aβ),Aβ是通过淀粉样前体蛋白(amyloid precursor protein,APP)的蛋白裂解产生。众多证据表明线粒体的功能异常对AD的发病起到了关键作用。体内和体外研究支持线粒体在APP代谢和细胞转运中起的重要作用。近年来,作为线粒体转录终止因子家族成员之一的线粒体转录终止因子4(mitochondrial transcription termination factor 4,MTERF4)被发现具有调节线粒体DNA转录和直接控制线粒体核糖体翻译的功能。MTERF4可能通过对线粒体功能的调节因而和AD的发病相关。目的:本研究观察MTERF4在APP/PS1双转基因小鼠海马组织中的表达情况,进一步研究MTERF4过表达与敲低在体外对APP代谢途径的影响与机制,揭示MTERF4在AD发病机制中的作用。方法:利用Western blot技术检测MTERF4在6月龄APP/PS1转基因小鼠中及同月龄野生对照小鼠海马组织中的表达情况。用分子克隆技术,构建pc DNA-MTERF4真核表达载体和p GPH1/GFP/Neo-MTERF4干扰载体。培养HEK293-APPswe细胞,分别将构建的过表达载体和RNA干扰载体转染细胞,采用Western blot技术检测细胞中MTERF4、APP、C99、C83、ADAM10和BACE1的蛋白表达水平。用Trizol法提取细胞的总RNA,反转录合成c DNA后进行实时荧光定量PCR检测,观察MTERF4、APP、ADAM10和BACE1基因在转录水平上的表达。构建含有ADAM10启动子序列的荧光素酶报告基因质粒p GL3-ADAM10,将pc DNA-MTERF4与p GL3-ADAM10共转染到HEK293细胞中,用海肾荧光素酶活性作为内参,通过对重组质粒表达荧光素酶活性的分析,确定过表达MTERF4对ADAM10基因启动子活性的影响。应用酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)来检测分泌到细胞外的Aβ42的生成量。采用流式细胞技术检测MTERF4过表达与敲低时的细胞周期情况。CCK-8法检测MTERF4过表达与敲低对HEK293-APPswe细胞增殖的影响。结果:Western blot结果显示在APP/PS1双转基因小鼠的海马组织中MTERF4蛋白表达水平增加68%。成功构建针对MTERF4基因的pc DNA-MTERF4过表达载体和p GPH1/GFP/Neo-MTERF4干扰载体。与对照组相比,将重组质粒pc DNA-MTERF4瞬时转染到HEK293-APPswe细胞24和48小时,MTERF4蛋白水平分别显著增加了67%和258%。实时定量PCR结果显示,pc DNA-MTERF4转染的细胞中MTERF4 m RNA水平在24和48小时增加235和216倍。MTERF4的过表达诱导了HEK293-APPswe细胞中APP蛋白和胞外分泌的Aβ42水平的显著增加。另外,APP蛋白切割加工产生的C99水平升高37%、C83水平降低27%,表明MTERF4过表达抑制了APP蛋白的α-切割而促进了β切割。实时荧光定量PCR结果显示,MTERF4过表达组细胞中的ADAM10 m RNA水平在转染48小时后降低68%。Western blot检测显示MTERF4的过表达抑制ADAM10的蛋白表达(下降27%)。双荧光素酶报告基因检测结果显示过表达MTERF4蛋白可抑制ADAM10基因启动子区域的启动子活性。MTERF4过表达不影响HEK293-APPswe细胞的周期分布和细胞增殖。在HEK293-APPswe细胞中利用p GPH1/GFP/Neo-MTERF4质粒干扰MTERF4基因的表达。将重组质粒转染到HEK293-APPswe细胞24和48小时,MTERF4蛋白水平分别降低了9%和36%。定量实时PCR结果显示,MTERF4敲低的细胞中MTERF4 m RNA水平在24和48小时降低19%和66%。与对照组相比,敲低MTERF4 48小时后细胞中APP蛋白表达水平降低26%,胞外Aβ42水平降低24%。同时APP代谢产物C99和C83水平分别降低19%和32%。干扰质粒转染细胞48h后,细胞中ADAM10蛋白水平降低了26%,而在24小时细胞中ADAM10蛋白水平无明显变化。与对照组相比,在MTERF4敲低24和48小时后,细胞中ADAM10 m RNA表达分别降低21%和51%。敲低MTERF4 24和48小时,细胞周期在G0/G1期的细胞百分比减少、S期细胞百分比增加,G2/M期细胞百分比无明显差异。干扰质粒转染细胞48h后,与对照组相比较细胞增殖降低11%。结论:MTERF4蛋白表达在APP/PS1转基因小鼠海马组织中上调。在体外该基因过表达增加了HEK293-APPswe细胞中APP蛋白水平并通过抑制α-分泌酶ADAM10促进APP的淀粉样蛋白形成加工。敲低MTERF4表达则通过抑制细胞中APP表达降低淀粉样蛋白形成加工,并对APP和ADAM10基因在转录和翻译水平都具有下调作用。另外,敲低MTERF4表达对细胞周期和细胞增殖具有一定的影响。这些结果表明MTERF4对引发AD的APP表达及代谢通路具有调控作用,两者呈正相关关系。MTERF4可能在AD的发病机制中发挥重要作用,该机制为探索AD的治疗方法提供了新思路。
[Abstract]:Background: Alzheimer's disease (Alzheimer 's disease, AD) is a central nervous system degenerative disease in the elderly with progressive cognitive impairment, memory impairment, and personality behavior changes,.AD, which seriously affects the patient's living ability, severe patients and even daily life can not take care of themselves, to the family and to the family. The typical social burden of the whole society,.AD, is a typical feature of the beta amyloid protein (beta -Amyloid peptide, A beta) deposited in the brain, and A beta is produced by the protein lysis of the amyloid precursor protein (amyloid precursor protein, APP). Many evidence suggests that the dysfunction of the grain body plays a key role in the pathogenesis of AD. Internal and external studies support the important role of mitochondria in APP metabolism and cell transport. In recent years, mitochondrial transcription terminator 4 (mitochondrial transcription termination factor 4, MTERF4), one of the members of the mitochondrial transcription terminator family, has been found to regulate mitochondrial DNA transcription and direct control of the mitochondrial nucleus. The function of.MTERF4 may be related to the regulation of mitochondrial function and the pathogenesis of AD. Objective: To observe the expression of MTERF4 in the hippocampus of APP/PS1 double transgenic mice, and to further study the effect and mechanism of MTERF4 overexpression and knock low on the metabolic pathway of APP in vitro, and reveal the mechanism of MTERF4 in the pathogenesis of AD. Methods: Western blot technique was used to detect the expression of MTERF4 in the hippocampus of 6 month old APP/PS1 transgenic mice and the same month old wild control mice. Using molecular cloning technology, the eukaryotic expression vector of PC DNA-MTERF4 and the P GPH1/GFP/Neo-MTERF4 interference carrier were constructed. The overexpression of the constructed HEK293-APPswe cells was cultured respectively. The expression level of MTERF4, APP, C99, C83, ADAM10 and BACE1 in the cells was detected by the vector and the RNA interference carrier, and the total RNA of the cells was extracted by Trizol method. The luciferase reporter gene plasmid P GL3-ADAM10 of the ADAM10 promoter sequence, PC DNA-MTERF4 and P GL3-ADAM10 were co transfected into HEK293 cells. The activity of luciferase activity was used as the internal parameter. The effect of the expression of luciferase activity on the recombinant plasmid was analyzed to determine the effect of overexpression of MTERF4 on the activity of ADAM10 gene promoter. Enzyme-linked immunosorbent assay (ELISA) was used to detect the production of A beta 42 secreted outside the cell. Flow cytometry was used to detect the effect of MTERF4 over expression and the cell cycle when the knockout was low. The effect of MTERF4 overexpression and knock low on the proliferation of HEK293-APPswe cells was detected by the flow cytometry. Results: Western blot results showed in APP/PS. The expression level of MTERF4 protein in the hippocampus of 1 transgenic mice increased by 68%. and successfully constructed PC DNA-MTERF4 overexpression vector for MTERF4 gene and P GPH1/GFP/Neo-MTERF4 interference carrier. Compared with the control group, the recombinant plasmid PC DNA-MTERF4 was transiently transfected to HEK293-APPswe cells for 24 and 48 hours, and the MTERF4 protein level was significantly increased. The results of 67% and 258%. real-time quantitative PCR showed that the MTERF4 m RNA level in PC DNA-MTERF4 transfected cells increased by 235 and 216 times of.MTERF4, which induced a significant increase in APP protein and extracellular secretion of A beta 42 in HEK293-APPswe cells. Moreover, the level of APP protein cutting increased by 37%. Decrease of 27%, indicating that MTERF4 overexpression inhibits the alpha cutting of APP protein and promotes beta cutting. Real-time fluorescence quantitative PCR results show that ADAM10 m RNA levels in MTERF4 overexpressed cells decrease 68%.Western blot detection after 48 hours transfection, indicating that the overexpression of MTERF4 inhibits ADAM10 protein expression (decreased by 27%). The results showed that the overexpression of MTERF4 protein inhibited the overexpression of promoter activity of the promoter region of the ADAM10 gene, which did not affect the cycle distribution and cell proliferation of HEK293-APPswe cells. The P GPH1/GFP/Neo-MTERF4 plasmid was used to interfere with the expression of MTERF4 gene in HEK293-APPswe cells. The recombinant plasmid was transfected to HEK293-APPswe fine. At 24 and 48 hours, the MTERF4 protein level decreased by 9% and 36%. quantitative real-time PCR results showed that the MTERF4 m RNA level in the MTERF4 knockout cells decreased 19% and 66%. was 19% and 66%. compared to the control group, and the expression level of APP protein in the cells decreased by 26% after knocking down MTERF4 48 hours, and the A beta 42 level decreased at the same time. The level of ADAM10 protein in the cells decreased by 26% and the level of ADAM10 protein was not significantly changed in the 24 hours after the 83 level decreased by 19% and 32%. interference plasmid transfected cells. Compared with the control group, the expression of ADAM10 m RNA in the cells decreased by 24 and 48 hours, and the ADAM10 m RNA expression decreased by 21% and 51%. knockdown MTERF4 24 and 48 hours, and the cell cycle of the cell cycle was 24 and 48 hours, respectively. The percentage of cells in the G0/G1 phase decreased, the percentage of S cells increased, and there was no significant difference in the percentage of G2/M cells. After the transfection of plasmid 48h, the cell proliferation decreased with the control group 11%. conclusion: the expression of MTERF4 protein was up-regulated in the hippocampus of APP/PS1 transgenic mice. The gene overexpression increased HEK293-APPswe fines in vitro. The level of APP protein in the cell and the inhibition of the alpha secretase ADAM10 promotes the formation of amyloid in APP. The low MTERF4 expression reduces the formation of amyloid by inhibiting the expression of APP in the cells, and has a downregulation effect on both the transcription and translation levels of the APP and ADAM10 genes. The proliferation has a certain effect. These results suggest that MTERF4 has a regulatory effect on the APP expression and metabolic pathways that lead to AD. The positive correlation between the two is that.MTERF4 may play an important role in the pathogenesis of AD. This mechanism provides a new way of thinking for the exploration of the treatment of AD.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16

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