【作者】 张璐；

【导师】 马腾；

【作者基本信息】 中国医科大学 ， 神经生物学， 2023， 硕士

【摘要】 目的:血脑屏障（blood-brain barrier,BBB）是内皮细胞的高度选择性和半渗透性边界,将脑组织与周围环境隔离开来。BBB中的内皮细胞通过紧密连接（tight junction,TJ）支架蛋白的偶联形成屏障,从而作为血管稳态的保护屏障。小鼠研究显示,TJ蛋白缺乏会导致BBB渗漏。血脑屏障通透性增高已被确定为阿尔茨海默症（Alzheimer’s disease,AD）发病机制的早期征兆。因此,血脑屏障的完整性与AD病程高度相关,在治疗或预防AD发病时,维持最佳血脑屏障功能非常重要。转录因子（transcription factors,TF）在内皮细胞中的作用与TJ蛋白表达高度相关。有研究发现转录因子ZNF787的过度表达对神经元发育有负面影响。虽然这些研究表明ZNF787与神经系统的结构和功能相关,但其在AD微环境中BBB中的作用仍不明确。此外,多项研究表明,HDAC1在血管内皮细胞和血源性内皮细胞中起重要作用,这表明HDAC1对内皮细胞的功能很重要。鉴于这些证据,我们推测HDAC1可能对调节AD背景下的BBB通透性改变有重要意义。生物信息学研究预测ZNF787和HDAC1之间的蛋白质相互作用,这种相互作用极有可能与BBB功能和屏障通透性有关。因此,我们研究了AD环境下的BBB中ZNF787和HDAC1的表达变化以及二者的互相作用对BBB通透性的影响,为AD的预防和治疗提供新思路。研究方法:共培养人星形胶质细胞和内皮细胞以建立体外BBB模型（ECs）,然后用Aβ_1-42孵育内皮细胞以模拟AD微环境。q RT-RCR和Western blot对ZNF787和HDAC1的内源性表达以及TJ相关蛋白的表达进行了检测。构建ZNF787和HDAC1的表达沉默载体,转染内皮细胞,研究ZNF787和HDAC1在内皮细胞中的作用和对BBB结构与功能的影响。TEER值测定和HRP通量测定检测BBB通透性。免疫荧光检测TJ相关蛋白的分布。Ch IP实验和双荧光素酶报告基因实验研究ZNF787对TJ蛋白的转录调控作用,Co-IP实验研究ZNF787和HDAC1的蛋白质相互作用。结果:ZNF787和HDAC1在Aβ孵育的ECs中高表达,沉默ZNF787诱导高TEER值与低HRP渗透率,TJ蛋白高表达。ZNF787与TJ蛋白的启动子区结合,抑制TJ蛋白的转录。沉默ZNF787诱导HDAC1低表达,沉默HDAC1诱导ZNF787低表达,共沉默ZNF787和HDAC1进一步诱导高TEER值与低HRP渗透率,TJ蛋白高表达,Co-IP实验显示ZNF787和HDAC1存在蛋白质相互作用。结论:1.Aβ_1-42孵育的ECs中ZNF787高表达,与BBB通透性增加相关。2.转录因子ZNF787可以与claudin-5和occludin基因的启动子区结合,抑制TJ蛋白的表达从而提高BBB的通透性。3.Aβ_1-42孵育的ECs中HDAC1高表达,与BBB通透性增加相关。4.ZNF787和HDAC1蛋白质相互作用调控BBB的通透性。更多还原

【Abstract】 Objective:The blood-brain barrier（BBB）is a highly selective and semi-permeable border of endothelial cells that separates the brain tissue from the peripheral environment.Endothelial cells in the BBB form a barrier through coupling of tight junction（TJ）scaffold proteins so as serve as a protective barrier for blood vessel homeostasis.Studies in mice have shown that the lack of TJ proteins can result in BBB leakage.In the context of Alzheimer’s disease（AD）,increased permeability of the BBB has been identified as an early marker of disease pathogenesis.Therefore,BBB integrity is highly relevant to AD,and interventions that maintain optimal BBB function are important to consider when treating or preventing the onset of AD.The role of transcription factors（TF）in endothelial cells is highly relevant to the gelationon of TJ protein expression.Kutsche and colleagues found that overexpression of ZNF787 negatively influenced neuronal development.While these studies indicate that ZNF787 is relevant to the structure and function of the nervous system,its roles in the BBB in the AD microenvironment remain poorly characterized.Furthermore,multiple studies demonstrate that HDAC1 plays important roles in vascular endothelial cells and blood-derived endothelial cells,altogether suggesting that HDAC1 is important for the functions of endothelial cells.Given these lines of evidence,we speculated that HDAC1 might be important for regulating BBB permeability in the context of AD.Furthermore,bioinformatic studies predict protein-protein interaction between ZNF787 and HDAC1,and this interaction may even be relevant to BBB functions and barrier permeability.Therefore,we investigated the expression of ZNF787 and HDAC1 in BBB of AD environment and the effect of their interaction on the permeability of BBB,hoping to provide new ideas for the prevention and treatment of AD.Methods:Human astrocytes and endothelial cells were co-cultured to establish an in vitro BBB model（ECs）,and endothelial cells were incubated with Aβ_1-42 to simulate the AD microenvironment.The endogenous expressions of ZNF787 and HDAC1 and the expression of TJ-related proteins were detected by q RT-RCR and Western blot.The silencing vectors of ZNF787 and HDAC1 were constructed and transfected into endothelial cells to study the effect of ZNF787 and HDAC1 on the structure and function of BBB.The permeability of BBB was detected by TEER assay and HRP flux assay.The distribution of TJ-related proteins was detected by immunofluorescence.The transcriptional regulation of ZNF787 on TJ protein was studied by Ch IP assay and double luciferase reporter assay,and the interaction between ZNF787 and HDAC1 protein was studied by Co-IP assay.Results:ZNF787 and HDAC1 was highly expressed in Aβ_1-42-incubated ECs,silencing ZNF787 induced high TEER value and low HRP permeability,as well as high expression of TJ protein.ZNF787 binds to the promoter region of TJ protein and inhibits the transcription of TJ protein.Silencing ZNF787 induced low expression of HDAC1,silencing HDAC1 induced low expression of ZNF787,and down-regulation of ZNF787and HDAC1 furtherly induced high TEER value and low HRP permeability,as well as high expression of TJ protein.Co-IP assay showed protein interaction between ZNF787and HDAC1.Conclusion:1.Elevated levels of ZNF787 in ECs incubated with Aβ_1-42 correlate with increased permeability of the BBB.2.Transcription factor ZNF787 can bind to the promoter region of claudin-5 and occludin genes,inhibiting the expression of TJ protein and thereby improving the permeability of BBB.3.HDAC1 was highly expressed in ECs incubated with Aβ_1-42,which correlated with increased BBB permeability.4.Protein-protein interaction between ZNF787 and HDAC1 mediates the permeability of the BBB.更多还原