Understand and Improve Iron Distribution and Erythropoiesis in Beta-Thalassemia

描述（由申请人提供）：本申请描述了一个为期五年的指导培训计划，以发展一个独立的临床医生，科学家，其职业生涯在学术医学将侧重于进一步我们对人类红细胞发育的理解。人类β-地中海贫血是一种与贫血、脾肿大和无效红细胞生成相关的疾病;这种疾病中的铁过载是由于输血和铁调素缺乏引起的铁吸收增加。我们的实验室先前证明，在患有β-地中海贫血的小鼠中用转铁蛋白治疗改善了疾病表型，逆转了贫血、脾肿大和无效的红细胞生成，并增加了铁调素表达。令人惊讶的是，尽管循环中有更多的红细胞，但它们的大小和血红蛋白含量在治疗小鼠中减少。我们推测，补充转铁蛋白导致更多的缺铁红细胞生成，减少血红素，α-珠蛋白，β-珠蛋白合成β地中海贫血之间的不平衡。我为这个奖项提出的研究目标是测试这个假设。我计划两个具体的目标来评估这一点：1）以确定铁的吸收红细胞前体是否改变时，额外的转铁蛋白是在体内和体外;和2）以确定如果调节和生产血红蛋白成分以及铁和血红素输出改变时，补充转铁蛋白被添加到正常和β-地中海贫血细胞在体内和体外。这些研究将验证我的假设，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，将提供对β地中海贫血表型生理学的深入了解，并将进一步加深我们对正常和异常红细胞生成的理解。本研究将通过以下方法实现这些具体目标：1）在给予额外的转铁蛋白的小鼠中表征红系前体细胞的铁动力学，分析这些细胞中转铁蛋白受体的体内表达，并评估细胞系和培养的人细胞中胞浆铁摄取; 2）评估血红素和球蛋白产生的表达水平和调节剂，测量胞质溶胶中锌原卟啉和游离血红素的水平，并表征在小鼠骨髓和脾样品以及培养的人样品中红系前体分化期间分别对FLVCR和FPN-1B、血红素和铁输出体的影响。这些研究侧重于基本的红细胞生理学，并可能有助于了解红细胞发育受到干扰的人类疾病。最后，在理解转铁蛋白在β地中海贫血小鼠中的功效机制方面取得的进展可能使其能够为患有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.