AMPO模型中RPE细胞β淀粉样蛋白表达增高的表观遗传机制研究

发布时间：2019-01-18 08:08

【摘要】：目的年龄相关性黄斑变性(age-related macular degeneration,AMD)是老年人群中心视力丧失的重要原因。其干性亚型所占比例高且目前仍未有有效治疗方式。玻璃膜疣(drusen)是其“Clinical hallmark”。β淀粉样蛋白(amyloid Aβ)是drusen重要组成成分,其在AMD中代谢异常机制未知。课题组前期Meta分析结果显示AMD患者血同型半胱氨酸含量升高,提示DNA甲基化异常可能参与了AMD的发生发展。本研究在建立A2E介导的光氧化应激(A2E-meadiated photooxidation,AMPO)模型的基础上探索视网膜色素上皮(retinal pigment epithelial,RPE)细胞β淀粉样蛋白表达增高的表观遗传机制。材料与方法采用MTS法检测AMPO模型及对照组中不同浓度A2E及造模后不同时间点ARPE-19细胞的细胞活力。采用流式细胞仪技术检测RPE细胞内ROS水平(DHE法)。采用Elisa试剂盒检测ARPE-19细胞上清Aβ1-40及Aβ1-42的含量。采用Real-time PCR以及Western blot技术检测Aβ生成相关蛋白APP,β-,γ-裂解酶APP、BACE1、PS1以及表观遗传调控酶系DNA甲基转移酶(DNA methyltransferase,DNMT),甲基化结合蛋白MECP2及组蛋白去乙酰化酶HDAC1 m RNA及蛋白的表达含量。采用Maldi-Tof MS技术对BACE1基因启动子区域397个bp长度(-388至2)位点进行甲基化测序。结果MTS结果显示,AMPO模型中随着时间的延长,细胞活力逐渐降低(P0.05)。6h时,随着A2E浓度的增加(6.25μM,12.5μM,25μM,50μM)细胞活力逐渐降低(P0.05)。且随着A2E浓度增加,DHE探针荧光强度在阈值内的比例比增高,提示细胞内ROS水平上升,与对照组相比均有明显差异(P0.05)。Elisa结果显示细胞上清中Aβ1-40及Aβ1-42的含量较对照组均有明显升高(P0.05),且抗氧化剂NAC(20m M)能抑制光氧化应激后产生的Aβ1-40及Aβ1-42分泌增加(P0.05)。造模组BACE1 m RNA及蛋白表达水平较对照组明显升高(P0.05),而APP及PS1 m RNA表达水平未见明显变化(P0.05)。甲基转移酶DNMT1、DNMT3a的含量较对照组均有明显的下降(P0.05)。Maldi-Tof结果显示造模后BACE1基因启动子区域397个bp长度的区域(-388至2)位点13,14-15,16-18的甲基化程度下降(P0.05),而其余位点甲基化程度无显著差异。结论在AMPO模型中,光氧化应激引起甲基转移酶表达减少,BACE1基因启动子特异性位点去甲基化,转录增强,β裂解效率增高,Aβ生成增多。提示了AMPO模型中表观遗传机制调控了RPE细胞分泌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 -, 纬 -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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774

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