發(fā)布時(shí)間：2019-01-18 08:08

【摘要】：目的年齡相關(guān)性黃斑變性(age-related macular degeneration,AMD)是老年人群中心視力喪失的重要原因。其干性亞型所占比例高且目前仍未有有效治療方式。玻璃膜疣(drusen)是其“Clinical hallmark”。β淀粉樣蛋白(amyloid Aβ)是drusen重要組成成分,其在AMD中代謝異常機(jī)制未知。課題組前期Meta分析結(jié)果顯示AMD患者血同型半胱氨酸含量升高,提示DNA甲基化異�？赡軈⑴c了AMD的發(fā)生發(fā)展。本研究在建立A2E介導(dǎo)的光氧化應(yīng)激(A2E-meadiated photooxidation,AMPO)模型的基礎(chǔ)上探索視網(wǎng)膜色素上皮(retinal pigment epithelial,RPE)細(xì)胞β淀粉樣蛋白表達(dá)增高的表觀(guān)遺傳機(jī)制。材料與方法采用MTS法檢測(cè)AMPO模型及對(duì)照組中不同濃度A2E及造模后不同時(shí)間點(diǎn)ARPE-19細(xì)胞的細(xì)胞活力。采用流式細(xì)胞儀技術(shù)檢測(cè)RPE細(xì)胞內(nèi)ROS水平(DHE法)。采用Elisa試劑盒檢測(cè)ARPE-19細(xì)胞上清Aβ1-40及Aβ1-42的含量。采用Real-time PCR以及Western blot技術(shù)檢測(cè)Aβ生成相關(guān)蛋白APP,β-,γ-裂解酶APP、BACE1、PS1以及表觀(guān)遺傳調(diào)控酶系DNA甲基轉(zhuǎn)移酶(DNA methyltransferase,DNMT),甲基化結(jié)合蛋白MECP2及組蛋白去乙�；窰DAC1 m RNA及蛋白的表達(dá)含量。采用Maldi-Tof MS技術(shù)對(duì)BACE1基因啟動(dòng)子區(qū)域397個(gè)bp長(zhǎng)度(-388至2)位點(diǎn)進(jìn)行甲基化測(cè)序。結(jié)果MTS結(jié)果顯示,AMPO模型中隨著時(shí)間的延長(zhǎng),細(xì)胞活力逐漸降低(P0.05)。6h時(shí),隨著A2E濃度的增加(6.25μM,12.5μM,25μM,50μM)細(xì)胞活力逐漸降低(P0.05)。且隨著A2E濃度增加,DHE探針熒光強(qiáng)度在閾值內(nèi)的比例比增高,提示細(xì)胞內(nèi)ROS水平上升,與對(duì)照組相比均有明顯差異(P0.05)。Elisa結(jié)果顯示細(xì)胞上清中Aβ1-40及Aβ1-42的含量較對(duì)照組均有明顯升高(P0.05),且抗氧化劑NAC(20m M)能抑制光氧化應(yīng)激后產(chǎn)生的Aβ1-40及Aβ1-42分泌增加(P0.05)。造模組BACE1 m RNA及蛋白表達(dá)水平較對(duì)照組明顯升高(P0.05),而APP及PS1 m RNA表達(dá)水平未見(jiàn)明顯變化(P0.05)。甲基轉(zhuǎn)移酶DNMT1、DNMT3a的含量較對(duì)照組均有明顯的下降(P0.05)。Maldi-Tof結(jié)果顯示造模后BACE1基因啟動(dòng)子區(qū)域397個(gè)bp長(zhǎng)度的區(qū)域(-388至2)位點(diǎn)13,14-15,16-18的甲基化程度下降(P0.05),而其余位點(diǎn)甲基化程度無(wú)顯著差異。結(jié)論在AMPO模型中,光氧化應(yīng)激引起甲基轉(zhuǎn)移酶表達(dá)減少,BACE1基因啟動(dòng)子特異性位點(diǎn)去甲基化,轉(zhuǎn)錄增強(qiáng),β裂解效率增高,Aβ生成增多。提示了AMPO模型中表觀(guān)遺傳機(jī)制調(diào)控了RPE細(xì)胞分泌Aβ增多。
[Abstract]:Objective Age-related macular degeneration (age-related macular degeneration,AMD) is an important cause of central vision loss in the elderly. Its dry subtype accounts for a high proportion and there is no effective treatment. (drusen) is an important component of Clinical hallmark. 尾 amyloid protein (amyloid A 尾 is an important component of drusen. The mechanism of abnormal metabolism in AMD is unknown. The results of Meta analysis showed that the level of homocysteine increased in patients with AMD, suggesting that abnormal DNA methylation might be involved in the occurrence and development of AMD. In this study, the epigenetic mechanism of increased 尾 -amyloid protein expression in retinal pigment epithelium (retinal pigment epithelial,RPE) cells was explored on the basis of A2E-mediated photooxidative stress (A2E-meadiated photooxidation,AMPO) model. Materials and methods MTS assay was used to detect the cell viability of ARPE-19 cells in AMPO model and control group at different concentrations of A2E and at different time points after modeling. The level of ROS in RPE cells was detected by flow cytometry (DHE). The levels of A 尾 1 40 and A 尾 1 42 in supernatant of ARPE-19 cells were detected by Elisa kit. APP, 尾 -, 緯 -lyase APP,BACE1,PS1 and DNA methyltransferase (DNA methyltransferase,DNMT) were detected by Real-time PCR and Western blot. Methylation binding protein MECP2, histone deacetylase HDAC1 m RNA and protein expression. A total of 397 bp sites (-388 to 2) in the promoter region of the BACE1 gene were sequenced by Maldi-Tof MS. Results MTS showed that the cell viability in AMPO model decreased with time (P0.05) and decreased with the increase of A2E concentration (6.25 渭 M 12.5 渭 M 25 渭 M 50 渭 M) (P0.05). With the increase of A2E concentration, the ratio of fluorescence intensity of DHE probe to threshold value increased, suggesting that the level of ROS in cells increased. The levels of A 尾 1-40 and A 尾 1-42 in the supernatant were significantly higher than those in the control group (P0.05), the results of). Elisa showed that the contents of A 尾 1-40 and A 尾 1-42 in the supernatant were significantly higher than those in the control group (P0.05). The antioxidant NAC (20mm) inhibited the increase of A 尾 1-40 and A 尾 1-42 secretion after photooxidative stress (P0.05). The expression of BACE1 m RNA and protein in the model group was significantly higher than that in the control group (P0.05), while the expression of APP and PS1 m RNA did not change significantly (P0.05). Methyltransferase DNMT1, The content of DNMT3a was significantly lower than that of the control group (P0.05). The results of Maldi-Tof showed that the methylation degree of 1314-1516-18 in the promoter region of BACE1 gene (-388 to 2) decreased significantly (P0.05). However, there was no significant difference in the degree of methylation at other sites. Conclusion in AMPO model, photooxidative stress induced decreased methyltransferase expression, BACE1 promoter specific site demethylation, enhanced transcription, increased 尾 cleavage efficiency and increased A 尾 production. The results suggest that the epigenetic mechanism in AMPO model regulates the increase of A 尾 secretion by RPE cells.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R774

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 祖衡兵,陳生弟,邵鈞,梁梁,徐潔懿;β淀粉樣蛋白與白細(xì)胞介素-1α表達(dá)的相關(guān)性研究[J];中國(guó)老年學(xué)雜志;2001年01期

2 羅南萍,楊道理,陳青,彭立義,孫曉明,劉桂香;β淀粉樣蛋白與生長(zhǎng)因子變化與老年性癡呆致病因素的研究[J];腦與神經(jīng)疾病雜志;2001年02期

3 武一,吉尚戎,蔣伍玲,隋森芳;β淀粉樣蛋白1-40和1-42的聚集性質(zhì)比較[J];電子顯微學(xué)報(bào);2003年01期

4 林煜,陳俊拋,賀順龍,蘇愛(ài)武;地塞米松對(duì)β淀粉樣蛋白誘導(dǎo)的腦內(nèi)炎癥損傷的保護(hù)作用[J];臨床神經(jīng)病學(xué)雜志;2004年01期

5 劉元;華茜;雷洪濤;李澎濤;;老年斑β淀粉樣蛋白的清除機(jī)制[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2008年01期

6 王麗芳;鄧湘平;;β淀粉樣蛋白對(duì)大鼠腎上腺嗜鉻瘤細(xì)胞的毒性作用[J];中國(guó)醫(yī)療前沿;2009年14期

7 熊坤林;楊清武;熊任平;;人β淀粉樣蛋白1-42在大腸桿菌中的表達(dá)純化及鑒定[J];重慶醫(yī)學(xué);2010年03期

8 張榮超;付于;于建春;韓景獻(xiàn);;β淀粉樣蛋白致線(xiàn)粒體功能障礙的研究現(xiàn)狀[J];中國(guó)老年學(xué)雜志;2010年15期

9 王芳;周愛(ài)民;;β淀粉樣蛋白的研究進(jìn)展[J];醫(yī)學(xué)綜述;2012年20期

10 祖衡兵;β淀粉樣蛋白受體研究進(jìn)展[J];國(guó)外醫(yī)學(xué).神經(jīng)病學(xué)神經(jīng)外科學(xué)分冊(cè);1998年06期

相關(guān)會(huì)議論文 前10條

1 史偉雄;汪洋;羅玉敏;洪震;;腦內(nèi)β淀粉樣蛋白_(1-40)經(jīng)血腦屏障外流轉(zhuǎn)運(yùn)的實(shí)驗(yàn)研究[A];第九次全國(guó)神經(jīng)病學(xué)學(xué)術(shù)大會(huì)論文匯編[C];2006年

2 秦偉;賈建平;;早老素1V97L突變對(duì)β淀粉樣蛋白降解酶的影響[A];第十一屆全國(guó)神經(jīng)病學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2008年

3 王乃東;;大鼠短暫性前腦不全缺血再灌注后β淀粉樣蛋白在海馬區(qū)的表達(dá)[A];中華醫(yī)學(xué)會(huì)第七次全國(guó)神經(jīng)病學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2004年

4 李慧貞;;測(cè)定老年癡呆β淀粉樣蛋白對(duì)臨床及醫(yī)學(xué)研究的價(jià)值[A];福建省第十次核醫(yī)學(xué)學(xué)術(shù)會(huì)議論文摘要匯編[C];2005年

5 葛金芳;喬金平;甘昌勝;潘見(jiàn);汪琛瑋;齊叢叢;蔡莉;周江寧;;抗β淀粉樣蛋白聚集化合物的體外篩選[A];Proceedings of the 8th Biennial Conference of the Chinese Society for Neuroscience[C];2009年

6 周輝;盛柏楊;徐葛林;嚴(yán)家新;;抗β淀粉樣蛋白(Aβ)單克隆抗體對(duì)老年癡呆模型細(xì)胞的影響[A];2011中國(guó)生物制品年會(huì)暨第十一次全國(guó)生物制品學(xué)術(shù)研討會(huì)論文集[C];2011年

7 蔣與剛;房恒通;龐偉;房紅蕓;;β淀粉樣蛋白處理大鼠認(rèn)知和微量營(yíng)養(yǎng)素狀況的變化[A];中國(guó)營(yíng)養(yǎng)學(xué)會(huì)論文匯編之二[C];2007年

8 高玲煥;鄒莉波;遲天燕;;β淀粉樣蛋白在阿爾茨海默病病因?qū)W中作用研究進(jìn)展[A];兩岸三地藥理學(xué)與臨床藥理學(xué)術(shù)會(huì)議論文集[C];2008年

9 汪琴;孫建國(guó);吳婷;;有氧運(yùn)動(dòng)對(duì)β淀粉樣蛋白25-35誘導(dǎo)的小鼠學(xué)習(xí)記憶損傷的影響[A];華東六省一市第22屆解剖與組織胚胎學(xué)學(xué)術(shù)年會(huì)暨山東解剖學(xué)會(huì)成立60周年紀(jì)念大會(huì)論文匯編[C];2013年

10 張敏;郗文輝;王晨軒;楊延蓮;;來(lái)源于促腎上腺皮質(zhì)激素的新型β淀粉樣蛋白高親和力抑制劑[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第01分會(huì)：表面物理化學(xué)[C];2014年

相關(guān)重要報(bào)紙文章 前3條

1 ;老藥ｃｌｉｏｑｕｉｎｏｌ治老年癡呆[N];大眾科技報(bào);2002年

2 記者 藍(lán)建中;常見(jiàn)老年頑疾有望接種預(yù)防[N];新華每日電訊;2013年

3 本報(bào)特約撰稿人 樊平;AD領(lǐng)域：待挖的“金礦”[N];醫(yī)藥經(jīng)濟(jì)報(bào);2009年

相關(guān)博士學(xué)位論文 前10條

1 張永蘭;京尼平苷調(diào)節(jié)β淀粉樣蛋白神經(jīng)毒性及其代謝的分子機(jī)制[D];重慶大學(xué);2015年

2 葉冰;β淀粉樣蛋白對(duì)星型膠質(zhì)細(xì)胞ATP結(jié)合盒式轉(zhuǎn)運(yùn)蛋白的表達(dá)調(diào)控及膽固醇轉(zhuǎn)運(yùn)等功能改變?cè)诎柎暮Ｄ≈械淖饔肹D];福建醫(yī)科大學(xué);2014年

3 黃s釗，

本文編號(hào)：2410490