GENESIS OF DENSE SICKLE CELLS

这项提议的目的是要了解致密的起源。 (脱水)镰状红细胞(RBC)。已知致密细胞可使 对镰状细胞病的病理生理学做出了重要贡献， 但仍有许多问题尚未解决。特别是，目前还不清楚 为什么一些镰刀状的红细胞在从骨头中长出来后不久就变得致密 骨髓变得密集，而其他的变得缓慢或根本不密集。Hb F 似乎可以保护细胞不会迅速变得致密，而且 对Hb F增强疗法的研究使得理解 Hb F与致密细胞形成的关系具体目标 本研究的主要内容有：(1)测定致密物质的形成速度 体内的细胞，特别是那些正处于快车道上的细胞 迅速变得密集，(2)评估细胞因素，如Hb F 在体内调节细胞脱水速率的内容，(3)到 确定体内导致钾外流的途径 快速通道细胞的脱水，以及(4)确定镰状细胞是否 是体内形成致密细胞所必需的。大量的 关于密度限定的镰刀体外特性的信息 单元格可用，但这些数据很难解释 使用放射性同位素方法获得的年龄，这是不可能的 今天。拟议的研究利用两种新的非同位素技术来 研究年龄和密度定义的镰状红细胞。第一种是利用 新生镰刀上转铁蛋白受体(TFR)的存在 网织红细胞。这一标记使研究非常年轻的年龄成为可能- 在每个密度分数中匹配的细胞数量。广泛的研究 将比较轻和重TFR+细胞的钾通道 以确定几条候选路径中的哪一条可能负责 “快速通道”牢房。此外，TFR+细胞将从 用免疫磁性技术定向的密度定义的组分 抗TFR，并测定其HbF含量。第二种新技术 是回输少量富含网织红细胞的生物素 清淡的红细胞。它们表面的生物素允许随后的定量 通过流式细胞术和链霉亲和素从循环中分离- 涂覆的磁珠。这些细胞将被用来跟踪依赖于时间的 体内变化，包括致密物质的形成和清除速度 细胞。

The goal of this proposal is to understand the genesis of dense (dehydrated) sickle red blood cells (RBC). Dense cells are known to make an important contribution to the pathophysiology of sickle cell disease, but a number of issues remain unresolved. In particular, it is not clear why some sickle RBC become dense soon after emerging from the bone marrow, while others become dense slowly or perhaps not at all. Hb F appears to protect cells from becoming dense quickly, and the emergence of Hb F-augmenting therapies makes it important to understand the relationship between Hb F and dense cell formation. The specific aims of this research are (1) to determine the rate of formation of dense cells in vivo, particularly those which are on the "fast track" toward becoming dense quickly, (2) to evaluate the cellular factors, e.g. Hb F content, which modulate the rate of cellular dehydration in vivo, (3) to determine the potassium efflux pathways which leads to in vivo dehydration of "fast track" cells, and (4) to determine whether sickling is a requirement for dense cell formation in vivo. A large amount of information concerning the in vitro properties of density-defined sickle cells is available, but these data are difficult to interpret due to the age obtained, using radioisotopic methods which would not be possible today. The proposed studies utilize two new non-isotopic techniques for studying age-and density-defined sickle RBC. The first takes advantage of the presence of transferrin receptors (TfR) on newly emergent sickle reticulocytes. This marker makes it possible to study a very young, age- matched population of cells in each density fraction. Extensive studies will compare the potassium flux pathways of light and heavy TfR+cells in order to determine which of several candidate pathways may be responsible for "fast track" cells. Furthermore, TfR+cells will be isolated from density-defined fractions with an immunomagnetic technique directed against TfR, and their Hb F content measured. The second novel technique is the reinfusion of a small volume of biotinylated, reticulocyte-rich light RBC. The biotin on their surface allows subsequent quantitation by flow cytometry and isolation from the circulation with streptavidin- coated magnetic beads. These cells will be used to follow time-dependent in vivo changes, including the rates of formation and removal of dense cells.