摘要： 目的 探讨长链非编码RNA-MEG8对阿尔茨海默病(AD)微环境下血脑屏障通透性的影响及作用机制。方法 应用β淀粉样蛋白(Aβ)处理人脑微血管内皮细胞hCMEC/D3(AD-ECs),模拟体外AD微环境下血脑屏障。实时荧光定量PCR检测正常人脑微血管内皮细胞(ECs)和AD-ECs中MEG8的表达。稳定转染沉默MEG8质粒后,应用微孔电阻系统检测跨内皮阻抗(TEER),TMB显色法测量辣根过氧化物酶(HRP)渗出量。免疫荧光法比较对照组、转染MEG8阴性对照组(sh-NC组)、转染MEG8沉默质粒组(sh-MEG8组)紧密连接相关蛋白ZO-1和occludin的分布变化,Western blotting检测ZO-1和occludin的表达。结果 MEG8在ADECs中的表达显著高于ECs(P<0.01)。与sh-NC组比较,sh-MEG8组TEER值显著升高,HRP渗出量显著降低(均P<0.01)。紧密连接相关蛋白ZO-1和occludin在AD-ECs边界呈现相对连续的分布。与sh-NC组比较,sh-MEG8组紧密连接相关蛋白ZO-1和occludin的表达显著增加(均P<0.01)。结论 MEG8在AD-ECs中表达上调,沉默MEG8表达显著降低AD微环境下血脑屏障的通透性,其作用机制可能是通过改变ZO-1和occludin的细胞分布,增加ZO-1和occludin的蛋白表达实现的。

MEG8对阿尔茨海默病微环境下血脑屏障通透性的影响及其作用机制

黄鑫¹, 朱璐², 林梅青³, 王继蕊³, 商秀丽³

1. 中国医科大学 附属第一医院放射科, 沈阳 110001;
2. 中国医科大学 附属盛京医院康复中心, 沈阳 110136;
3. 中国医科大学 附属第一医院神经内科, 沈阳 110001

收稿日期:2022-06-29出版日期:2022-12-30发布日期:2022-12-12
通讯作者:商秀丽E-mail:shang_zhao@sohu.com
作者简介:黄鑫 (1993-)，女，医师，硕士研究生.
基金资助:国家自然科学基金(81871104，82001485)；辽宁省科技厅科学技术计划(2017225027)；沈阳市科学技术计划(20-205-4-047)


关键词: MEG8, 阿尔茨海默病, 血脑屏障, 通透性
Abstract: Objective To investigate the role and potential mechanism of long non-coding RNA，MEG8 on the permeability of the blood-brain barrier in Alzheimer disease(AD) microenvironment. Methods β-amyloid protein(Aβ)-incubated brain microvascular endothelial cell line hCMEC/D3(provided by Dr. Couraud from Institut Cochin， Paris， France) was established to simulate the bloodbrain barrier in AD microenvironment in vitro. Real-time quantitative PCR(qRT-PCR) was used to detect the expression levels of MEG8 in human brain microvascular endothelial cells(ECs) and Aβ-incubated human brain microvascular endothelial cells(AD-ECs). After knock down of MEG8， the micropore resistance system was applied to measure the transendothelial electrical resistance(TEER)， and the TMB chromogenic test was conducted to assess the horseradish peroxidase(HRP) flux. In the control， sh-NC， and sh-MEG83 groups， the distribution and expression of tight junction-associated proteins ZO-1， occludin， and claudin-5 were detected by immunofluorescence assays and Western blotting， respectively. Results The expression level of MEG8 was significantly increased in AD-ECs(P<0.01). Compared with the sh-NC group， the TEER value of sh-MEG8 group was significantly increased， and HRP flux was significantly decreased (P<0.01). The distribution of tight-junction protein ZO-1 and occludin was continuous on cell boundaries. The expression levels of ZO-1 and occludin were significantly increased in the sh-MEG8 group compared with those in the sh-NC group(P<0.01). Conclusion The expression of MEG8 is upregulated in AD-ECs. The deletion of MEG8 significantly reduces the permeability of the blood-brain barrier in AD microenvironment， possibly via alternating the cellular distribution of ZO-1 and occludin and increasing the expression of ZO-1 and occludin.
Key words: MEG8, Alzheimer disease, blood-brain barrier, permeability
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