Functional Genomics of the Blood-Brain Barrier

描述（由申请人提供）： 艾滋病毒脑炎和艾滋病痴呆综合征是神经系统疾病，即使在高度活跃的逆转录病毒疗法的存在下，这些疾病仍然困扰着艾滋病患者。研究表明，乙醇可能会加剧这些症状，并可能通过调节血脑屏障（BBB）对HIV感染的单核细胞或游离HIV病毒浸润的敏感性来实现。为了研究这些复杂的相互作用，需要一种准确代表体内BBB的体外模型。然而，当脑微血管内皮细胞在体外培养时，它们经历大量的去分化并且失去了在体内观察到的许多专门的生化和形态特征。拟议的研究的总体目标是获得一个复杂的基因组范围内的了解体内血脑屏障表型的决定因素，在体外丢失，并朝着真正代表性的体外血脑屏障模型，通过重建体内环境的进展。 功能基因组学的抑制消减杂交的形式将被用来阐明完整的脑微血管（BBB）和原代培养的脑微血管内皮细胞之间的差异基因表达谱。这将允许鉴定引起独特的体内BBB表型的已知和新基因的功能簇。有了这个全球功能概况，基因微阵列将被创建并用作体外模型重建体内条件能力的定量评估的诊断。血管周围脑细胞与脑微血管内皮细胞体外共培养可恢复血脑屏障的某些特性。将使用诊断基因微阵列评估星形胶质细胞、神经元和周细胞对体外表型的贡献。将鉴定通过共培养重建的分子途径和生理特征，并为体外模型的合理调整提供靶点。最后，乙醇对体外模型的功能特征的影响将被研究作为用于研究HIV、乙醇和BBB之间复杂相互作用的体外模型的初步验证。

DESCRIPTION (provided by applicant): HIV encephalitis and AIDS dementia complex are neurological disorders that continue to afflict AIDS patients even in the presence of highly active retroviral therapies. Research has suggested that ethanol may exacerbate these symptoms and may do so by modulating the susceptibility of the blood-brain barrier (BBB) to infiltration by HIV infected monocytes or free HIV virus. In order to study these complex interactions, an in vitro model that accurately represents the in vivo BBB is required. When brain microvascular endothelial cell are cultured in vitro, however, they undergo a great deal of de-differentiation and lose many of the specialized biochemical and morphological features observed in vivo. The overall goal of the proposed research is to gain a sophisticated genome-wide understanding of the determinants of the in vivo BBB phenotype that are lost in vitro and progress towards truly representative in vitro BBB models by recreating the in vivo environment. Functional genomics in the form of suppression subtractive hybridization will be used to elucidate the differential gene expression profiles between intact brain microvessels (BBB) and primary cultures of brain microvessel endothelial cells. This will allow the identification of functional clusters of known and novel genes that elicit the unique in vivo BBB phenotype. Armed with this global functional profile, a gene microarray will be created and used as a diagnostic for the quantitative assessment of an in vitro model's ability to reestablish in vivo conditions. Perivascular brain cells can restore some BBB properties when co-cultured with brain microvascular endothelial cells in vitro. The contributions of astrocytes, neurons, and pericytes on the in vitro phenotype will be assessed using the diagnostic gene microarray. Molecular pathways and physiological characteristics that are reestablished by co-culture will be identified and provide targets for rational tuning of in vitro models. Finally, the effects of ethanol on the functional features of the in vitro model will be investigated as a preliminary validation of in vitro models for the study of the complicated interactions between HIV, ethanol, and the BBB.