FOLATE-RESPONSIVE DYSGENESIS

拟议研究的目的是证明 叶酸受体（FR）在调节母体转移中的中心作用 叶酸穿过胎盘和正常胚胎形成。调控异常 FR可能被确定为发育中胚胎发育不良的原因， 特别是神经管缺陷和神经畸形的产生 嵴细胞由于这些细胞经历增殖的爆发， FR调节的叶酸向胎盘的转移对他们的发育至关重要。 发展提出了五个具体目标。首先，PI将确定 FR在小鼠神经嵴细胞中的表达是否与爆发相关， 通过a）展示增殖停滞的效果， 与抗叶酸剂甲氨蝶呤联合，间隔12小时;和B） 通过免疫组化将这些结果与FR表达的变化相关联 和原位杂交。其次，PI将确定体内叶酸是否 缺乏可导致妊娠当天胎盘和组织中FR的上调 17使用叶酸缺乏或叶酸充足的饮食。FR表达方法 将包括北方和西方印迹研究 转录和翻译后事件以及核运行研究 叶酸缺乏症将通过叶酸监测， 同型半胱氨酸测量。将通过以下方法评价胎仔组织： 免疫组织化学和原位杂交。第三，一系列实验 以确定是否持续淬火胎盘FR期间， 使用反义FR的母体叶酸缺乏将不利地影响胎盘 增殖并导致胎儿生长迟缓。这些研究将使用 脂质体工程化以递送并入胎盘中的反义FR cDNA 特异性启动子在两个时间点，妊娠第8天和第16天。第四，他的 研究小组将确定特定的人类43-kDa分子是否与 在FR的5 '-UTR中具有顺式元件的反式因子肝蛋白是 与小鼠模型相同。这些研究将涉及隔离和 从小鼠胎盘中纯化反式因子蛋白， 使用特异性抗体表征其功能。他们将扩展 以前的观察表明，同型半胱氨酸（Hcy）是一个调解人的反-顺 互动这些实验将使用凝胶迁移试验， 切片和大浓度（500-100 mM）的Hcy。第五，小组将 测量离体小鼠胎儿中顺式和反式元件的相互作用 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，并确定其表达与FR的一致性， 小鼠胚胎组织。此外，他们还将评估下调 顺式和反式反义寡核苷酸的FR表达 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.