Organotypic Culture as a Model for EBV Infection of Epithelial Cells

描述（由申请人提供）：eb病毒（EBV）与许多B淋巴细胞和上皮源性恶性肿瘤相关。其中许多是艾滋病患者特有的或增加的，包括头颈部的上皮癌。本研究的重点是利用器官型培养建立eb病毒感染上皮细胞的体外模型。器官型培养与单层培养的不同之处在于细胞分化和分层，类似于原位上皮。由于eb病毒可以在体内分化的上皮细胞中进行复制，因此器官型培养是研究上皮细胞在病毒生命周期中的作用的理想系统。我们的初步数据表明，我们可以使用表达GFP的重组EBV作为标记物，在培养中有效地感染原代口腔角化细胞，并且我们可以产生有效模拟口腔粘膜上皮的原代口腔角化细胞的器官型培养物。基于这些初步数据，我们提出在器官型培养中鉴定感染的细胞和表达的病毒基因产物来表征EBV感染。几项研究表明EBV可能在体内建立潜伏感染。我们将确定eb病毒是否可以建立潜伏感染并确定潜伏类型。我们将研究病毒基因产物在上皮细胞中表达的功能。因此，我们将在R21资助机制的有限范围内，建立并表征EBV在上皮内研究的模型系统，并利用该系统尽可能明确病毒基因产物的功能。这些研究将为未来的实验奠定基础，这些实验将检验关于EBV感染人类口腔上皮的几个长期存在的问题。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is associated with a number of malignancies of B lymphocyte and epithelial origin. Many of these are specific to or increased in AIDS patients including epithelial cancers of the head and neck. This proposal focuses on the development of an in vitro model of EBV infection of epithelium using organotypic cultures. Organotypic cultures differ from monolayers in that the cells differentiate and stratify to resemble epithelium found in situ. Because EBV can replicate in differentiated epithelial cells in vivo, organotypic culture is an ideal system to study the role of epithelial cells in the viral life cycle. Our preliminary data demonstrate that we can efficiently infect primary oral keratinocytes in culture using a recombinant EBV that expresses GFP as a marker, and that we can generate organotypic cultures of primary oral keratinocytes that effectively mimic oral mucosal epithelium. Based on these preliminary data, we propose to characterize EBV infection in organotypic culture identifying the cells infected and the viral gene products expressed. Several studies have suggested that EBV may establish a latent infection in vivo. We will determine whether EBV can establish a latent infection and characterize the type of latency. The functions of the viral gene products expressed in epithelium will be examined. Thus, we will establish and characterize a model system for the study of EBV in epithelium and use this system to define the functions of the viral gene products as far as possible within the limited scope of the R21 funding mechanism. These studies will set the stage for future experiments that will examine several long standing questions concerning EBV infection of human oral epithelium. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is associated with a variety of human malignancies that occur predominantly in B lymphocytes or epithelial cells, many of which have an increased incidence in AIDS patients. The proposed research is intended to develop an in vitro model of EBV infection of epithelium that will be invaluable in filling in the gaps in our knowledge about the role of epithelial cells in the EBV lifecycle. Because the oral mucosa is the site of entry and exit of the virus, epithelial cells play a major role in viral spread and thus, understanding their role in the viral life cycle will not only shed light on this important step but has the potential to impact all downstream events from EBV infection. This model is likely to be important not only in adding to our knowledge of the role of the epithelium in the viral life cycle and the contribution of the virus to the development of malignancies, but could also be used for the development and/or testing of antiviral strategies.