Novel 3-D Model of Placental Cytomegalovirus Infection

描述（由申请人提供）：本提案的总体目标是开发一种改进的组织模型，用于研究妊娠期间人类巨细胞病毒感染（CMV）。具体来说，将利用旋转壁容器（RWVs）来建立这一先进的模型系统。巨细胞病毒是先天性病毒感染的主要原因，大约50%的原发性巨细胞病毒感染孕妇发生宫内传播。妊娠期巨细胞病毒感染不仅会对新生儿造成灾难性后果，还会影响妊娠结局，包括自然流产、宫内生长受限和先兆子痫。尽管先天性巨细胞病毒感染有显著的发病率和死亡率，但人们对这种病毒如何感染胎儿知之甚少。RWV技术提供了一种以前未应用于胎盘组织研究的独特方法，并为探索宫内巨细胞病毒感染提供了一种新的模型系统。这些生物反应器的培养条件提供了低剪切和低湍流的生长环境，类似于正常的体内胎儿和胎盘发育所需的环境。这样的环境允许细胞在空间上共定位，三维生长，并分化成高度特化的组织。本研究提出的初步数据表明，RWV支持妊娠早期细胞滋养细胞系的生长。重要的是，我们已经证明，在生物反应器内创造的环境促进细胞滋养层分化，并允许形成复杂的多层三维（3-D）聚集体，其功能类似于子宫滋养层细胞。本研究的假设是，在RWV中培养的胎盘上皮细胞在功能上与子宫胎盘中的发育相似，这些细胞的三维生长将为研究宫内巨细胞病毒感染的分子细节提供一个改进的模型系统。本课题的长期研究目标是利用三维模型研究和了解1)正常胎盘发育的分子细节，2)巨细胞病毒的发病机制，3)妊娠期间母胎界面发生的宿主-病原体相互作用，以及4)巨细胞病毒对胎盘和妊娠结局影响的调节分子机制。具体目的是：1)进一步表征人类胎盘的三维模型；2)将胎盘三维模型应用于宫内CMV感染的研究。这种新的模型系统将有助于人类胎盘的深入研究，最终导致预防和治疗胎盘和先天性巨细胞病毒感染的新治疗策略。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop an improved tissue model for the study of human cytomegalovirus infection (CMV) during pregnancy. Specifically, rotating wall vessels (RWVs) will be utilized to establish this advanced model system. CMV is the leading cause of congenital viral infection, and intrauterine transmission of the virus occurs in approximately 50% of pregnant women with primary CMV infection. CMV infection during pregnancy can have catastrophic consequences not only on the neonate, but also on pregnancy outcome, including spontaneous abortion, intrauterine growth restriction and pre-eclampsia. Despite the significant morbidity and mortality associated with congenital CMV infection, little is known about how the virus infects the conceptus. RWV technology offers a unique approach not previously applied toward the study of placental tissues and provides a novel model system to explore intrauterine CMV infection. The culture conditions of these bioreactors provide a low-shear and low-turbulence growth environment, similar to that required for normal in vivo fetal and placental development. Such an environment allows cells to co-localize spatially, grow three-dimensionally, and differentiate into highly specialized tissues. Preliminary data presented in this proposal demonstrate that the RWV supports the growth of a first trimester cytotrophoblast cell line. Importantly, we have shown that the environment created within the bioreactor promotes cytotrophoblast differentiation and allows for the formation of complex, multi-layered three-dimensional (3-D) aggregates that are functionally similar to in utero trophoblast cells. The hypothesis to be tested within this proposal is that placental epithelial cells cultured in the RWV are functionally similar to the developing in utero placenta and that the 3-D growth of these cells will provide an improved model system for the study of the molecular details involved in intrauterine CMV infection. The long-term research goals of this proposal are to utilize the 3-D model to study and understand 1) the molecular details of normal placental development, 2) CMV pathogenesis, 3) host-pathogen interactions that occur at the maternal-fetal interface during pregnancy, and 4) the molecular mechanism(s) that regulate the effect of CMV on placentation and pregnancy outcome. The specific aims are: 1) to further characterize the 3-D model of human placentation, and 2) to apply the placental 3-D model toward the study of intrauterine CMV infection. This novel model system will contribute to advanced studies of human placentation, ultimately leading to new therapeutic strategies for the prevention and treatment of placental and congenital CMV infection.