Understand and Improve Iron Distribution and Erythropoiesis in Beta-Thalassemia

描述(由申请者提供)：本申请描述了一项为期五年的导师培训计划，旨在培养一名独立的临床医生和科学家，他的学术医学生涯将专注于加深我们对人类红细胞发育的理解。人类中的β-地中海贫血是一种与贫血、脾肿大和无效的红细胞生成有关的疾病；这种疾病的铁超载是由于输血和缺乏海普西丁引起的铁吸收增加所致。我们的实验室以前证明，用转铁蛋白治疗β-地中海贫血小鼠可以逆转贫血、脾肿大、无效的红细胞生成和海普西丁表达增加的疾病表型。令人惊讶的是，尽管有更多的红细胞在循环中，但它们的大小和血红蛋白含量在接受治疗的小鼠中减少了。我们假设补充转铁蛋白会导致更多缺铁的红细胞生成，从而减少β-地中海贫血患者血红素、α-珠蛋白和β-珠蛋白合成的失衡。我在这一奖项建议的研究中的目标是检验这一假设。我计划有两个具体的目标来评估这一点：1)确定当体内和体外有额外的转铁蛋白时，红系前体细胞的铁摄取是否发生变化；2)确定在体内和体外添加补充转铁蛋白时，正常和β-地中海贫血细胞中血红蛋白成分的调节和产生以及铁和血红素的输出是否发生改变。这些研究将检验我的假设，即外源性转铁蛋白限制铁进入红系前体细胞并保护早期红系细胞免受血红素毒性，将有助于深入了解β-地中海贫血表型的生理机制，并将加深我们对正常和紊乱的红细胞生成的理解。我将通过下列方法达到这些特定目标：1)表征给予额外转铁蛋白的小鼠红系前体的铁动力学，分析这些细胞中转铁蛋白受体的表达，并评估细胞系和培养的人细胞中铁的摄取；2)评估血红素和珠蛋白的表达水平和调节，检测细胞质中锌原卟啉和游离血红素的水平，并表征在红系前体细胞分化过程中，分别对FLVCR和FPN-1B、血红素和铁出口商的影响。这些研究侧重于基本的红细胞生理学，并可能有助于深入了解红细胞发育受到干扰的人类疾病。最后，对转铁蛋白在β-地中海贫血小鼠中疗效机制的了解取得的进展可能使其能够为这种疾病的患者开发治疗方法。

DESCRIPTION (provided by applicant): This application describes a five-year mentored training program to develop an independent clinician-scientist whose career in academic medicine will focus on furthering our understanding of human red blood cell development. Beta-thalassemia in humans is a disease associated with anemia, splenomegaly, and ineffective erythropoiesis; iron overload in this disease results from transfusion and increased iron absorption from hepcidin deficiency. Our lab previously demonstrated that treatment with transferrin in mice with beta- thalassemia improves the disease phenotype with reversal of anemia, splenomegaly, and ineffective erythropoiesis and an increase in hepcidin expression. Surprisingly, although there are more red cells in circulation, their size and hemoglobin content is reduced in treated mice. We hypothesize that supplemental transferrin results in more iron deficient erythropoiesis, reducing the imbalance between heme, alpha-globin, and beta-globin synthesis in beta-thalassemia. My goal in the studies proposed for this award is to test this hypothesis. I plan two specific aims to evaluate this: 1) to determine if iron uptake by erythroid precursors is altered when additional transferrin is available in vivo and in vitro; and 2) to determine if the regulation and production of hemoglobin components as well as iron and heme export is altered when supplemental transferrin is added in normal and beta-thalassemic cells in vivo and in vitro. These studies will test my hypothesis that exogenous transferrin restricts iron entry into erythroid precursors and protects early erythroid cells from heme toxicity, will provide insight into the physiology responsible for the phenotype in beta- thalassemia, and will further our understanding of normal and disordered erythropoiesis. I will accomplish these specific aims by the following methods: 1) characterize the ferrokinetics of erythroid precursors in mice given additional transferrin, analyze the expression of transferrin receptor in these cells in vivo, and evaluate cytosolic iron uptake in cell lines and human cells in culture; 2) evaluate expression levels and regulators of heme and globin production, measure levels of zinc protoporphyrin and free heme in the cytosol, and characterize the effect on FLVCR and FPN-1B, heme and iron exporters, respectively, during erythroid precursor differentiation in mouse bone marrow and spleen samples as well as in human samples in culture. These studies focus on basic red blood cell physiology and may lend insight into human diseases where red blood cell development is disturbed. Lastly, progress in understanding mechanisms of transferrin efficacy in beta-thalassemic mice may enable its therapeutic development for patients with this disease.