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症复合体是神经疾病，即使在存在高度有效的逆转录病毒疗法的情况下，也会继续困扰艾滋病患者。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)对感染艾滋病毒的单核细胞或游离艾滋病毒的渗透的敏感性来做到这一点。为了研究这些复杂的相互作用，需要一个能准确反映体内血脑屏障的体外模型。然而，当脑微血管内皮细胞在体外培养时，它们经历了大量的去分化，失去了体内观察到的许多特殊的生化和形态特征。这项拟议研究的总体目标是在基因组范围内对在体外丢失的体内BBB表型的决定因素进行复杂的了解，并通过重建体内环境向真正具有代表性的体外BBB模型迈进。 抑制消减杂交的功能基因组学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。这将允许识别引起体内独特的BBB表型的已知和新基因的功能簇。有了这一全球功能图谱，基因微阵列将被创建并用作定量评估体外模型重建体内条件的能力的诊断。血管周围脑细胞与脑微血管内皮细胞体外共培养可恢复部分血脑屏障特性。将使用诊断基因芯片评估星形胶质细胞、神经元和周细胞对体外表型的贡献。通过共培养重建的分子途径和生理特性将被识别，并为体外模型的合理调整提供靶点。最后，将研究乙醇对体外模型功能特征的影响，作为研究HIV、乙醇和血脑屏障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.