MECHANISMS CONTROLLING CELLULAR FETAL HEMOGLOBIN CONTENT

控制细胞胎儿血红蛋白含量的机制

基本信息

批准号：
2216213
负责人：
George J Dover
金额：
$ 37.73万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-01-01 至 1994-12-31
项目状态：
已结题

项目摘要

The purpose of this application is to delineate the mechanisms which control the cellular production of adult and fetal hemoglobin (HbA and HbF). Three principles underlie this proposal: 1) HbF is confined in adults to a subpopulation of red cells (F cells) which contain both HbF and HbA; 2) increases postnatally in HbF levels are primarily associated with increases in F cell production not increased HbF in all red cells; and 3) increases in F cell production ameliorates the clinical severity of sickle cell (SS) disease and certain forms of thalassemia. Our Specific Aims focus on three categories: First, how are F cells different from non-F cells? Using single cell immunoassays with monoclonal antibodies to human HbF the mean corpuscular hemoglobin, mean cell volume and concentration of HbS in F cells and non F cells in SS patients will be measured and the variables which control preferential F cell survival in SS disease will be determined. By autoradiographic and fluorescent flow cytometry techniques it will be determined whether F cells skip terminal cell division during maturataion and whether precursors of F cells have unique antigens (surface receptors) or different erythrocyte enzyme activities than non-F cells. Second, using comparisons between sibs with SS disease, the number of genetic loci which control F cell production and the genetic control of HbF levels in F cells will be analyzed. Having separated F erythroblasts from non-F erythroblasts using immunoadsorption techniques, differences in chromatin structure (DNAse I hypersensitive sites) and the methylation patterns of CpG dinucleotides around the Gamma-Delta-Beta-globin gene complex will be assessed. Third, the effect of cell-cycle specific agents (Hydroxyurea, 5-azacytidine) on increasing HbF production in severely affected SS patients will be studied. Five to 15 SS patients will be treated with varying doses of hydoxyurea to determine the rate of onset and duration of increased F cell production. Dose-response analyses of hydroxyurea versus % F cell production will be measured and the toxicity of drug regiments which maintain elevated F cell production levels will be monitored. The in vitro erythroid culture system will be used to analyze the effect of cell-cycle specific drugs on erythroid maturation and HbF production. The study of F cells can serve as a model for understanding how differentiation is controlled at the cellular level and how cell divisions affect differential gene expression.

此应用程序的目的是描述这些机制控制成人和胎儿血红蛋白的细胞产生（HBA和 HBF）。该提议的基础的三个原则：1）HBF被限制在成年人对既包含HBF和HBF的红细胞（F细胞）的亚群 HBA; 2）HBF水平的产后增加主要与在所有红细胞中，F细胞产生的增加并未增加HBF； 3） F细胞产生的增加可以缓解镰刀的临床严重程度细胞（SS）疾病和某些形式的丘脑贫血。我们的具体目标专注于三类：首先，F单元与非f的分支有何不同细胞？使用与人类单克隆抗体的单细胞免疫测定 HBF平均白细胞血红蛋白，平均细胞体积和浓度将测量F细胞中F细胞和非F细胞中的非F细胞，并测量控制SS病中优先F细胞存活的变量将是决定。通过放射自显影和荧光流式细胞仪技术将确定F细胞是否跳过末端细胞分裂成熟以及F细胞的前体是否具有独特的抗原（表面受体）或与非F细胞不同的红细胞酶活性。其次，使用SS疾病的SIB之间的比较，数量控制F细胞产生的遗传基因座和HBF的遗传控制 F细胞中的水平将进行分析。已经分离了f的生物细胞使用免疫吸附技术的非F生成细胞，差异染色质结构（DNase I超敏位点）和甲基化 CpG二核苷酸的模式周围的伽马 - 戴尔塔 - β-球蛋白基因复杂将评估。第三，细胞周期特异性药物的作用（羟基脲，5-氮杂丁胺）严重增加HBF的生产将研究受影响的SS患者。五到15例SS患者将是用不同剂量的羟基脲处理，以确定发病率 F细胞产生增加的持续时间。剂量反应分析将测量羟基脲与％F细胞的产生，并毒性的毒性维持F细胞生产水平升高的药物团将是受监控。体外红细胞培养系统将用于分析细胞周期特异性药物对红系成熟和HBF的影响生产。 F细胞的研究可以作为理解的模型如何在细胞水平以及细胞如何控制分化分裂影响差异基因表达。