FOLATE-RESPONSIVE DYSGENESIS

拟议研究的目的是证明 叶酸受体(FR)在母体转运调节中的中枢作用 穿过胎盘和正常胚胎发育的叶酸。不正常的调节 FR可能被确定为发育中的胚胎发育不全的原因 特别是神经管缺陷和神经畸形的产生 冠状细胞。由于这些细胞经历了激增的增殖， FR调节的叶酸转移到胎盘对胎盘是必不可少的。 发展。提出了五个具体目标。首先，PI将确定 小鼠神经脊细胞中FR的表达与爆震性反应是否相关 通过a)显示增殖受阻的影响来实现细胞增殖 与抗叶酸甲氨蝶呤联合应用，每隔12小时；和b) 这些结果与免疫组织化学中FR表达的变化相关 和原位杂交。其次，等电点将决定叶酸在体内是否 孕期缺乏症引起胎盘及组织中FR表达上调 17使用叶酸缺乏或叶酸充足的饮食。FR表达的途径 在胎盘和组织中将包括Northern和Western印迹进行研究 转录和翻译后事件以及核连续研究 转录因子FR的表达。叶酸缺乏症将通过叶酸和 同型半胱氨酸测量。胎儿组织将通过以下方式进行评估 免疫组织化学和原位杂交。第三，一系列实验 建议用来确定胎盘FR是否持续猝灭 使用反义FR的母亲叶酸缺乏将对胎盘产生不利影响 增殖并导致胎儿生长迟缓。这些研究将使用 胎盘携带反义FR基因的脂质体 在两个时间点，怀孕8天和16天的特定启动子。 该小组将确定特定的人类43-kDa是否相互作用 FR5‘-UTR区含有顺式元件的反式因子肝蛋白 在小鼠模型中是相同的。这些研究将涉及隔离和 小鼠胎盘及胎盘中反式因子蛋白的纯化 利用特异性抗体对其功能进行表征。他们将延长 以往的观察表明，同型半胱氨酸(Hcy)是反式顺式反应的媒介。 互动。这些实验将对胎盘进行两种凝胶移位分析。 切片和大浓度(500-100 mM)的同型半胱氨酸。第五，集团将 体外培养的小鼠胚胎中顺式和反式元件相互作用的研究 文化。这将通过一种复杂的分子克隆策略来实现 小鼠反式因子及其与受体表达一致性的测定 小鼠胎儿组织。他们还将评估下调监管的效果 顺式和反式反义寡核苷酸表达FR的研究 元素。

The objective of the proposed studies is to demonstrate the central role of the folate receptor (FR) in regulation of transfer of maternal folates across the placenta and of normal embryogenesis. Abnormal regulation of FR may be established as a cause for dysgenesis of the developing embryo, in particular the production of neural tube defects and abnormalities of neural crest cells. Since these cells undergo bursts of proliferation, the FR-regulated transfer of folate to the placenta is essential to their development. Five specific aims are proposed. First, the PI will determine whether the expression of FR in mouse neural crest cells correlate with bursts of cellular proliferation by a) demonstrating effects of arrested proliferation in conjunction with antifolate methotrexate at 12-hour intervals; and b) correlating these results with changes in FR expression by immunohistochemistry and in situ hybridization. Second, the PI will determine whether in vivo folate deficiency induces up regulation of FR in placenta and tissues on gestation day 17 using folate-deficient or folate-replete diets. Approaches to FR expression in the placenta and tissues will include Northern and Western blots to study transcriptional and post-translational events as well as nuclear run-on studies of transcription of FR. Folate deficiency will be monitored by folate and homocysteine measurements. Fetal tissues will be evaluated by immunohistochemistry and in situ hybridization. Third, a series of experiments are proposed to determine whether sustained quenching of placental FR during maternal folate deficiency using antisense FR will adversely affect placental proliferation and result in fetal growth retardation. These studies will use liposomes engineered to deliver antisense FR cDNA incorporated in a placenta specific promoter at two time points, gestational days 8 and 16. Fourthly, his group will determine whether the interaction of a specific human 43-kDa trans-factor liver protein with the cis-element in the 5'-UTR of FR is identical in the mouse model. These studies will involve isolation and purification of the trans-factor protein from mouse placenta and characterization of its function using specific antibody. They will extend previous observations that homocysteine (Hcy) is a mediator of the trans - cis interaction. These experiments will use both gel shift assays with placental slices and large (500-100 mM) concentrations of Hcy. Fifthly, the group will measure the interaction of the cis and trans elements in ex vivo mouse fetal culture. This will be achieved by a complex strategy of molecular cloning of the mouse trans factor and determining concordance of its expression with FR in mouse fetal tissues. Also they will evaluate the effect of down-regulation of FR expression by antisense oligonucleotides to both the cis and the trans elements.