Human Amnion Cell Model for Investigation of Amniotic Fluid Volume

描述（由申请人提供）：在美国，5-10%的妊娠发生羊水（AF）量减少，通常与母体和胎儿发病率增加相关，这会影响妊娠结局，并产生潜在的长期不良后果。这大大增加了对临床干预的需求，从而增加了产科管理的成本。大约50%的症状性羊水过少和羊水过多的病因不明。然而，尽管发病率高，仍然没有有效的治疗方法来纠正不适当的AF体积，从而提高新生儿存活率。这主要是由于缺乏对调节AF体积的机制以及最终导致AF体积过多或减少的异常原因的理解。关于这一主题的研究数量有限，主要是因为在没有适当的人类模型的情况下，难以设计关于相对未知和复杂的调节机制的研究。在实验动物中进行的研究已经产生了关于AF跨羊膜（转运的限速层）转运特征的大多数现有知识。然而，一个关键的问题仍然是这些动物数据对人类的适用性，以及使用动物模型进行人类生物学研究的相关性。为了解决这一缺陷，我们建议开发一个人类模型，用于研究AF容量调节。我们最近开发了一种体外绵羊羊膜细胞模型，用于研究AF跨羊膜细胞单层的转运，该模型易于进行实验操作。我们的目标是使这种细胞培养系统适用于人羊膜细胞的转运研究。本申请旨在研究这种体外人羊膜细胞模型用于研究正常妊娠和羊水过少和羊水过多中AF转运机制的有效性。在特定目标1中，将确定AF跨人羊膜细胞单层转运的主动和被动组分作为溶质分子量的函数;将研究小窝在主动跨细胞转运中的参与;将探索VEGF 165及其抑制亚型在调节主动转运中的作用;并阐明羊水过少和羊水过多时羊膜细胞转运特性的改变。在特定目的2中，将检查VEGF 165对人羊膜细胞中水通道水通道蛋白1、3和9的被动扩散的调节。将分析羊水过少和羊水过多的羊膜细胞中水通道蛋白通道的被动扩散是否改变。在特定目标3中，将在正常妊娠、羊水过少和羊水过多的羊膜细胞中研究VEGF 165作为介导活性小窝转胞吞的细胞信号传导事件中的调节因子的作用。总体而言，这些研究将揭示这种人羊膜细胞培养模型是否是研究人类妊娠中AF体积调节机制的合适模型，并测试使用该模型解读羊水过少和羊水过多中异常AF体积的病因学的可行性。 公共卫生相关性：本研究将建立人羊膜细胞模型，并对其进行表征，以研究人类妊娠中调节羊水量的机制。从使用该模型的研究中获得的知识将允许阐明羊水过少和羊水过多的病因，从而为设计羊水异常的管理方案和治疗范例提供科学依据。

DESCRIPTION (provided by applicant): Abnormalities in amniotic fluid (AF) volume occur in 5-10% of pregnancies in the US and are often associated with increased maternal and fetal morbidity that compromises pregnancy outcome with potential long term adverse consequences. This dramatically increases the need for clinical intervention and thus costs of obstetrical management. Approximately 50% of symptomatic oligohydramnios and polyhydramnios have unknown etiology. However despite the high incidence, there remains no effective therapy for correcting the inappropriate AF volume thereby improving neonatal survival. This is primarily due to the lack of understanding of the mechanisms that regulate AF volume and the causes of the abnormalities that ultimately lead to excessive or reduced AF volumes. The limited amount of research on this subject is largely because of the difficulties in designing studies on relatively unknown and complex regulatory mechanisms without an appropriate human model. Studies in experimental animals have generated most of the current knowledge on AF transport characteristics across the amnion, the rate-limiting layer for transport. However, a critical question remains as to the applicability of these animal data to the human, and the relevance of using animal models for investigations of human biology. To address this deficiency, we propose to develop a human model for studies of AF volume regulation. We have recently developed an in vitro ovine amnion cell model for the study of AF transport across amnion cell monolayers that are readily accessible to experimental manipulations. Our goal is to adapt this cell culture system for transport studies in human amnion cells. The present application is designed to investigate the validity of this in vitro human amnion cell model for studies of AF transport mechanisms in normal pregnancies and in oligohydramnios and polyhydramnios. In Specific Aim 1, the active and passive components of AF transport across human amnion cell monolayers as a function of solute molecular weight will be determined; the involvement of caveolae in active transcellular transport will be investigated; the role of VEGF165 and its inhibitory isoform in regulating active transport will be explored; and alterations in transport characteristics in amnion cells from oligohydramnios and polyhydramnios will be elucidated. In Specific Aim 2, the VEGF165 regulation of passive diffusion across water channels aquaporins 1, 3 and 9 in human amnion cells will be examined. Whether passive diffusion across aquaporin channels in amnion cells from oligohydramnios and polyhydramnios is altered will be analyzed. In Specific Aim 3, the role of VEGF165 as the regulatory factor in the cell signaling events that mediate active caveolar transcytosis will be investigated in amnion cells from normal pregnancy, and from oligohydramnios and polyhydramnios. Overall, the studies will reveal whether this human amnion cell culture model is a suitable model for the investigation of AF volume regulatory mechanisms in human pregnancy, and test the feasibility of using this model to decipher the etiology underlying abnormal AF volume in oligohydramnios and polyhydramnios. PUBLIC HEALTH RELEVANCE: The proposed studies will establish and characterize a human amnion cell model for the investigation of mechanisms mediating amniotic fluid volume regulation in human pregnancy. Knowledge gained from studies using this model will allow the elucidation of the etiology of oligohydramnios and polyhydramnios, and thus provide the scientific basis for designing management protocols and treatment paradigms for amniotic fluid abnormalities.