In Vivo Hydration Changes in Hemoglobin SS and SC Cells

为了制定有效的镰状细胞病治疗策略， 需要细胞病理生理学。进入血液循环后，镰状细胞经历了一系列的变化和选择过程，这些变化和选择过程深刻地影响了镰状细胞的倾向 以及它们的生存特征。本建议的重点是了解这些随时间变化的。体内研究将追踪少量高度纯化的光或致密的光。 循环中的镰状细胞，利用生物素标记。这种方法是我们实验室独有的， 在体内细胞老化过程中对多种细胞特性的逐个细胞分析。其中一 已经认识到水化状态的性质是至关重要的。现在 似乎单向脱水模型是不够的，镰状细胞 最终会再水化并具有高钠含量，可能继发于严重的 膜损伤初步的生物素标记研究表明，这些钠 负载的细胞具有极短的体内存活期。加上他们在 这些数据强烈支持这些细胞作为终末细胞的重要作用， 状态拟议的研究将包括审查形成机制， 这些年老的水合细胞的生存特征。镰状细胞的一个机理模型 再水化，基于转运抑制剂DIDS和布美他尼的初步研究， 将在拟议的研究中进行测试。相关的实验将检查非常高水平的磷脂酰丝氨酸（PS）外化，初步研究表明，在体外再水合的镰状细胞。体内和体外研究将评估非常年轻的红系细胞（镰状和非镰状）中存在的低水平PS暴露，以及随着镰状细胞老化和脱水而出现的高水平暴露。患有SC和CC疾病的患者提供了与纯合子镰状细胞病的重要且信息丰富的对照。脱水动力学和生存特性的SC和CC细胞将探索使用建立和新开发的实验方法。这将允许与SS细胞的类似研究进行比较，并导致更好地理解这些疾病脱水机制的相似性和差异。

To develop effective treatment strategies for sickle cell disease, a clear understanding of cellular pathophysiology is required. After entering the circulation, sickle cells experience a number of changes and selection processes that profoundly influence their propensity to sickle and their survival characteristics. This proposal is focused on understanding these time-dependent changes. In vivo studies will track a small volume of highly purified light or dense sickle cells in the circulation, utilizing a biotin label. This method, unique to our laboratory, offers a cell-by-cell analysis of multiple cellular properties during in vivo cell aging. One of these properties, hydration state, has been recognized to be of paramount importance. It now appears that a one-directional dehydration model is not adequate, and that sickle cells eventually become rehydrated and have high sodium content, perhaps secondary to severe membrane damage. A preliminary biotin label study has demonstrated that these sodium loaded cells have an extremely short in vivo survival. Taken together with their numbers in the circulation, these data strongly support an important role for these cells as a terminal cellular state. The proposed studies will include examinations of the mechanism of formation and survival characteristics of these older, hydrated cells. A mechanistic model of sickle cell rehydration, based on preliminary studies with the transport inhibitors DIDS and bumetanide, will be tested in the proposed studies. Related experiments will examine the remarkably high level of phosphatidylserine (PS) externalization that preliminary studies demonstrated in sickle cells that were rehydrated in vitro. In vivo and in vitro studies will evaluate both the low level PS exposure that is present in very young erythroid cells, both sickle and nonsickle, and the high level exposure that appears to develop as sickle cells age and become dehydrated. Patients with SC and CC disease offer an important and informative counterpoint to homozygous sickle cell disease. The dehydration kinetics and survival characteristics of SC and CC cells will be explored using established and newly developed experimental approaches. This will allow a comparison to similar investigations with SS cells, and lead to a better understanding of similarities and differences in the dehydration mechanisms for these diseases.