Project 4: The Placenta-specific Glucose Transporter Modulation: obesity, metabol

项目概要/摘要-项目4 肥胖症的增加，特别是在工业化国家，已成为流行病。在 美国三分之二的成年人（20岁及以上）超重或肥胖，体重指数或BMI 25 kg/m2以上。这种流行病蔓延到怀孕人口，其中超过一半的美国人 妇女进入怀孕与增加的体重指数，因此肥胖是一个最常见的 高危产科综合症母亲超重和肥胖与胎儿发育障碍有关 生长导致新生儿预后不良，易患心血管疾病和代谢性疾病 在以后的生活中出现紊乱。胎盘作为胎儿和母体循环之间的界面， 它的主要功能之一是为胎儿提供营养。由于胎儿可获得的营养量是 作为胎儿生长的重要决定因素，胎盘与胎儿过度生长有关。的机制 人们对子宫内营养过剩、胎儿过度生长和以后疾病发展之间的联系知之甚少 并被认为包括胎盘营养转运、遗传和表观遗传变化的改变。我们 我建议开发一种基因治疗方法，使用一种独特的piggyBac转座子来解决这个问题， 基于转染系统，在我们的研究所开发的第一阶段，这个COBRE。这些精灵 质粒能够有效地将基因导入宿主基因组。此外，这种非病毒方法 与基于病毒的系统相比，具有许多优点，包括低毒性和低免疫应答。我们 最近采用了一种微创体内方法（超声靶向微泡破坏， UTMD）用于递送GENIE质粒，实现报告基因在小鼠肝脏中的长期表达。 小鼠UTMD可以介导生物活性分子向超声可及靶器官的位点特异性递送 例如胎盘。具体地说，我们将首先尝试通过稳定表达Glut 1来敲低小鼠的Glut 1表达。 通过UTMD将shRNAmir构建体引入胎盘的基因组中。然后我们将确定 Glut 1基因敲低对胎盘葡萄糖转运能力的影响 综合征的后代。我们还将确定Glut 1基因敲低对原代细胞功能的影响。 人滋养层（PHT）细胞。由于胎盘是一种具有有限寿命的组织，对胎儿至关重要， 但出生后丢弃，它非常适合通过遗传来改善胎儿健康。 修饰，而不会引起与基因直接转移到宿主基因组中相关的问题。的 我们将在本申请中检验的假设是胎盘特异性的Glut 1表达减少， 随后减少葡萄糖转运进入胎盘将减弱胎儿过度生长和随后的 高脂饮食母鼠后代的代谢综合征。此外，胎盘基因转移可能有助于 作为减少胎儿过度生长的早期干预策略。

PROJECT SUMMARY/ABSTRACT - Project 4 The increase in obesity, particularly in industrialized countries has taken on epidemic proportions. In the US two-thirds of adults (20 years of age and older) are overweight or obese, with a body mass index or BMI above 25kg/m2. This epidemic extends to the pregnant population where more than half of all American women enter pregnancy with an increased BMI and as a consequence obesity is one of the most common high-risk obstetric syndromes. Maternal overweight and obesity are associated with disturbances in fetal growth leading to poor neonatal outcomes and a predisposition for cardiovascular disease and metabolic disorders later in life. The placenta serves as an interface between the fetal and maternal circulation and one of its key functions is to supply nutrients to the fetus. As the quantity of nutrients available to the fetus is a significant determinant of fetal growth, the placenta has been implicated in fetal overgrowth. The mechanisms linking in utero nutrient excess, fetal overgrowth and disease development later in life are poorly understood and are thought to include alterations in placental nutrient transport, genetic and epigenetic changes. We propose to develop a gene therapy approach to address this problem using a unique piggyBac transposon- based transfection system that was developed at our institute in Phase I of this COBRE. These GENIE plasmids are able to efficiently introduce genes into the host genome. Moreover, this non-viral approach presents many advantages over virus-based systems, including low toxicity and low immune response. We have recently employed a minimally invasive in vivo method (Ultrasound Targeted Microbubble Destruction, UTMD) for the delivery of GENIE plasmids achieving long-term expression of a reporter gene in the liver of mice. UTMD can mediate site-specific delivery of bioactive molecules to ultrasound-accessible target organs such as the placenta. Specifically, we will first attempt to knockdown the Glut1 expression in mice by stably introducing shRNAmir constructs into the genome of the placenta via UTMD. We will then determine the effects of Glut1 knockdown on placental glucose transport capacity on fetal growth and the metabolic syndrome in the offspring. We will also determine the effects of Glut1 knockdown on the function of primary human trophoblast (PHT) cells. Since the placenta is a tissue with a finite life span, essential for fetal development but discarded after birth, it is well suited for improving fetal wellbeing through genetic modifications, without causing problems associated with gene transfer directly into the host genome. The hypothesis that we will test in this application is that a placenta-specific reduction in Glut1 expression and consequent decrease of glucose transport into the placenta will attenuate fetal overgrowth and subsequent metabolic syndrome in offspring from dams fed a high-fat diet. Furthermore, placental gene transfer may serve as an early intervention strategy to reduce fetal overgrowth.