HPV in Differentiated Human Airway Epithelia In Vitro

体外分化的人气道上皮细胞中的 HPV

• 批准号: 6926090
• 负责人: John H Lee
• 金额: $ 19.22万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6926090
• 关键词: Adenoviridae; cell differentiation; clinical research; electron microscopy; flow cytometry; gene expression; genome; human papillomavirus; human tissue; immunocytochemistry; life cycle; pathologic process; respiratory epithelium; tissue /cell culture; transfection /expression vector; virulence; virus DNA; virus RNA; virus cytopathogenic effect; virus genetics; virus protein

DESCRIPTION (provided by applicant): The goal of this application is to develop Dr. John Lee into an independent physician/scientist. Drs. Welsh and Klingelhutz will assume responsibility as mentors to ensure success in his development. The heart of this proposal is intensive laboratory training in the pathogenesis of human papilloma virus (HPV) related airway disease. HPV infection of the aerodigestive tract results in papilloma formation and the development of squamous cell cancer, leading causes of dysphonia and aphonia. An understanding of disease pathogenesis has been limited by the lack of an in vitro model of differentiated human airway epithelia (HAE) and the inability to produce experimentally useful quantities of HPV virions. However, our lab now routinely produces in vitro models of well-differentiated HAE. Moreover, studies in keratinocytes have circumvented the lack of HPV virions by delivering the HPV genome by transfection. However, airway epithelia have marked differences from keratinocytes, and comparable transfection methods cannot be used in airway epithelia. Therefore, I developed a novel strategy to deliver an HPV genome inserted in a recombinant adenovirus. In preliminary studies, I demonstrated the feasibility of this approach by transferring the HPV genome with high efficiency to well-differentiated HAE. The HPV genome was excised from the vector and circularized. These results allow me to now ask the following questions about the interaction between HPV and HAE. 1) What factors influence the establishment of viral persistence in human airway epithelia? I will learn whether the HPV genome persists in HAE, and whether it exists as an episome or as integrated DNA. The data will also show us how epithelial differentiation influences the pattern of genome persistence. 2) Does the HPV genome alter the morphology or differentiation of human airway epithelia? Studies addressing this question will help us understand cellular changes associated with HPV-induced alterations of the epithelium in vivo. 3) Do human airway epithelia produce infectious HPV virions? The answer to this question will provide important insight into the factors involved in a productive viral infection. 4) What is the temporal and spatial pattern of HPV gene expression in differentiated human airway epithelia? An investigation of the timing, localization, and quantity of viral mRNA and protein expression will begin to show us how differentiation impacts the viral life cycle. These studies will provide new insight into HPV-related airway disease and should hasten the development of new treatments.

描述（由申请人提供）：本申请的目的是将John Lee博士发展成独立的医师/科学家。博士。威尔士和克林胡特（Klingelhutz）将担任导师的责任，以确保他的发展取得成功。该提案的核心是针对人乳头瘤病毒（HPV）相关气道疾病发病机理的强化实验室培训。 HPV感染了机修道，导致乳头状瘤的形成和鳞状细胞癌的发展，吞咽困难和吞噬的主要原因。缺乏分化人类气道上皮（HAE）的体外模型和无法生产实验有用的HPV病毒体的缺乏体外模型，因此对疾病发病机理的理解受到限制。但是，我们的实验室现在通常会产生分化良好的HAE的体外模型。此外，在角质形成细胞中的研究通过转染传递了HPV基因组来规避缺乏HPV病毒体。然而，气道上皮与角质形成细胞有明显的差异，在气道上皮中不能使用可比的转染方法。因此，我制定了一种新型策略，以提供重组腺病毒中插入的HPV基因组。 在初步研究中，我通过以高效率将HPV基因组传递到差异化的HAE来证明了这种方法的可行性。从载体中切除HPV基因组并循环。这些结果使我现在可以询问有关HPV和HAE之间相互作用的以下问题。 1）哪些因素会影响人类气道上皮的病毒持久性的建立？我将了解HPV基因组是否持续存在于HAE中，以及它是否作为沿着沿着偶发或集成的DNA存在。数据还将向我们展示上皮分化如何影响基因组持久性的模式。 2）HPV基因组会改变人类气道上皮的形态或分化吗？解决此问题的研究将有助于我们了解与HPV诱导的体内上皮变化相关的细胞变化。 3）人类气道上皮会产生感染性的HPV病毒体吗？这个问题的答案将为涉及生产性病毒感染所涉及的因素提供重要的见解。 4）在分化的人类气道上皮中，HPV基因表达的时间和空间模式是什么？对病毒mRNA和蛋白质表达的时间，定位和数量的研究将开始向我们展示分化如何影响病毒生命周期。这些研究将为与HPV相关的气道疾病提供新的见解，并应加速新疗法的发展。