本文選題：阿爾茨海默病 + 淀粉樣前體蛋白　； 參考：《重慶醫(yī)科大學》2017年博士論文

【摘要】：背景:阿爾茨海默病(Alzheimer’s disease,AD)是一種以進行性的認知障礙、記憶力減退和性格行為改變等癥狀為主的影響老年人的中樞神經(jīng)系統(tǒng)退行性疾病。AD由于嚴重影響患者的生活能力,重癥患者甚至日常生活不能自理,給患者家庭和整個社會帶來沉重的經(jīng)濟和社會負擔。AD的典型特征是在腦中沉積的β-淀粉樣蛋白(β-Amyloid peptide,Aβ),Aβ是通過淀粉樣前體蛋白(amyloid precursor protein,APP)的蛋白裂解產(chǎn)生。眾多證據(jù)表明線粒體的功能異常對AD的發(fā)病起到了關(guān)鍵作用。體內(nèi)和體外研究支持線粒體在APP代謝和細胞轉(zhuǎn)運中起的重要作用。近年來,作為線粒體轉(zhuǎn)錄終止因子家族成員之一的線粒體轉(zhuǎn)錄終止因子4(mitochondrial transcription termination factor 4,MTERF4)被發(fā)現(xiàn)具有調(diào)節(jié)線粒體DNA轉(zhuǎn)錄和直接控制線粒體核糖體翻譯的功能。MTERF4可能通過對線粒體功能的調(diào)節(jié)因而和AD的發(fā)病相關(guān)。目的:本研究觀察MTERF4在APP/PS1雙轉(zhuǎn)基因小鼠海馬組織中的表達情況,進一步研究MTERF4過表達與敲低在體外對APP代謝途徑的影響與機制,揭示MTERF4在AD發(fā)病機制中的作用。方法:利用Western blot技術(shù)檢測MTERF4在6月齡APP/PS1轉(zhuǎn)基因小鼠中及同月齡野生對照小鼠海馬組織中的表達情況。用分子克隆技術(shù),構(gòu)建pc DNA-MTERF4真核表達載體和p GPH1/GFP/Neo-MTERF4干擾載體。培養(yǎng)HEK293-APPswe細胞,分別將構(gòu)建的過表達載體和RNA干擾載體轉(zhuǎn)染細胞,采用Western blot技術(shù)檢測細胞中MTERF4、APP、C99、C83、ADAM10和BACE1的蛋白表達水平。用Trizol法提取細胞的總RNA,反轉(zhuǎn)錄合成c DNA后進行實時熒光定量PCR檢測,觀察MTERF4、APP、ADAM10和BACE1基因在轉(zhuǎn)錄水平上的表達。構(gòu)建含有ADAM10啟動子序列的熒光素酶報告基因質(zhì)粒p GL3-ADAM10,將pc DNA-MTERF4與p GL3-ADAM10共轉(zhuǎn)染到HEK293細胞中,用海腎熒光素酶活性作為內(nèi)參,通過對重組質(zhì)粒表達熒光素酶活性的分析,確定過表達MTERF4對ADAM10基因啟動子活性的影響。應(yīng)用酶聯(lián)免疫吸附實驗(enzyme-linked immunosorbent assay,ELISA)來檢測分泌到細胞外的Aβ42的生成量。采用流式細胞技術(shù)檢測MTERF4過表達與敲低時的細胞周期情況。CCK-8法檢測MTERF4過表達與敲低對HEK293-APPswe細胞增殖的影響。結(jié)果:Western blot結(jié)果顯示在APP/PS1雙轉(zhuǎn)基因小鼠的海馬組織中MTERF4蛋白表達水平增加68%。成功構(gòu)建針對MTERF4基因的pc DNA-MTERF4過表達載體和p GPH1/GFP/Neo-MTERF4干擾載體。與對照組相比,將重組質(zhì)粒pc DNA-MTERF4瞬時轉(zhuǎn)染到HEK293-APPswe細胞24和48小時,MTERF4蛋白水平分別顯著增加了67%和258%。實時定量PCR結(jié)果顯示,pc DNA-MTERF4轉(zhuǎn)染的細胞中MTERF4 m RNA水平在24和48小時增加235和216倍。MTERF4的過表達誘導了HEK293-APPswe細胞中APP蛋白和胞外分泌的Aβ42水平的顯著增加。另外,APP蛋白切割加工產(chǎn)生的C99水平升高37%、C83水平降低27%,表明MTERF4過表達抑制了APP蛋白的α-切割而促進了β切割。實時熒光定量PCR結(jié)果顯示,MTERF4過表達組細胞中的ADAM10 m RNA水平在轉(zhuǎn)染48小時后降低68%。Western blot檢測顯示MTERF4的過表達抑制ADAM10的蛋白表達(下降27%)。雙熒光素酶報告基因檢測結(jié)果顯示過表達MTERF4蛋白可抑制ADAM10基因啟動子區(qū)域的啟動子活性。MTERF4過表達不影響HEK293-APPswe細胞的周期分布和細胞增殖。在HEK293-APPswe細胞中利用p GPH1/GFP/Neo-MTERF4質(zhì)粒干擾MTERF4基因的表達。將重組質(zhì)粒轉(zhuǎn)染到HEK293-APPswe細胞24和48小時,MTERF4蛋白水平分別降低了9%和36%。定量實時PCR結(jié)果顯示,MTERF4敲低的細胞中MTERF4 m RNA水平在24和48小時降低19%和66%。與對照組相比,敲低MTERF4 48小時后細胞中APP蛋白表達水平降低26%,胞外Aβ42水平降低24%。同時APP代謝產(chǎn)物C99和C83水平分別降低19%和32%。干擾質(zhì)粒轉(zhuǎn)染細胞48h后,細胞中ADAM10蛋白水平降低了26%,而在24小時細胞中ADAM10蛋白水平無明顯變化。與對照組相比,在MTERF4敲低24和48小時后,細胞中ADAM10 m RNA表達分別降低21%和51%。敲低MTERF4 24和48小時,細胞周期在G0/G1期的細胞百分比減少、S期細胞百分比增加,G2/M期細胞百分比無明顯差異。干擾質(zhì)粒轉(zhuǎn)染細胞48h后,與對照組相比較細胞增殖降低11%。結(jié)論:MTERF4蛋白表達在APP/PS1轉(zhuǎn)基因小鼠海馬組織中上調(diào)。在體外該基因過表達增加了HEK293-APPswe細胞中APP蛋白水平并通過抑制α-分泌酶ADAM10促進APP的淀粉樣蛋白形成加工。敲低MTERF4表達則通過抑制細胞中APP表達降低淀粉樣蛋白形成加工,并對APP和ADAM10基因在轉(zhuǎn)錄和翻譯水平都具有下調(diào)作用。另外,敲低MTERF4表達對細胞周期和細胞增殖具有一定的影響。這些結(jié)果表明MTERF4對引發(fā)AD的APP表達及代謝通路具有調(diào)控作用,兩者呈正相關(guān)關(guān)系。MTERF4可能在AD的發(fā)病機制中發(fā)揮重要作用,該機制為探索AD的治療方法提供了新思路。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R749.16

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