In Vivo Hydration Changes in Hemoglobin SS and SC Cells

为镰状细胞病制定有效的治疗策略，明确认识 细胞病理生理学是必需的。进入循环后，镰状细胞经历了许多变化和选择过程，这些变化和选择过程深刻地影响了它们的镰状倾向。 以及它们的生存特征。这项提议的重点是理解这些依赖于时间的变化。体内研究将追踪少量高纯度的轻或密 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，它提供了 活体细胞老化过程中多种细胞特性的逐个细胞分析。这其中的一个 特性，即水合状态，已被认为是至关重要的。现在就是 看来，单向脱水模型是不够的，而镰状细胞 最终变得补水并有高钠含量，可能是继发性到重度 膜损伤。初步的生物素标记研究表明，这些钠 负载细胞在体内的存活时间极短。与他们在 循环，这些数据有力地支持了这些细胞作为终末细胞的重要作用 州政府。拟议的研究将包括对形成机制和 这些较老的水合细胞的生存特征。一种镰状细胞的力学模型 再水化，基于运输抑制剂DIDS和布美他尼的初步研究， 将在拟议的研究中进行测试。相关实验将检验在体外复水的镰状细胞中，初步研究证明磷脂酰丝氨酸(PS)的外化水平非常高。体内和体外研究将评估非常年轻的红系细胞中存在的低水平PS暴露，无论是镰状细胞还是非镰状细胞，以及随着镰状细胞老化和脱水而出现的高水平暴露。SC和CC疾病的患者提供了一个重要的和提供信息的纯合子镰状细胞疾病的对应物。将利用已建立的和新开发的实验方法来探索SC和CC细胞的脱水动力学和生存特性。这将允许与SS细胞的类似研究进行比较，并有助于更好地了解这些疾病脱水机制的异同。

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.