RHEOLOGICAL STUDIES OF SICKLE CELL DISEASE

拟议工作的目标是利用现代 流变学技术与生化分析相结合 了解镰刀病病理生理的生物物理基础 细胞性贫血。然后，这些知识将为 合理开发临床新疗法。具体来说， 将调查两大类问题：(1)红细胞- 可控流动条件下内皮细胞的相互作用 使用表观荧光视频显微镜和图像分析 (2)切应力对红细胞膜的影响 通透性和镰刀状血红蛋白聚合率和 最终的凝胶材料特性。结构性交互作用与 内皮细胞导致组织氧合和血管缺乏- 由于凝胶形成而导致的咬合是两个可能的问题 可能会导致临床危机。所提出的技术将 检查这些区域。调查需要非常专门的 设备--其中一些只有在我们的实验室里才有。 可控流动条件下红细胞的视频显微镜研究 条件，我们可以区分细胞是否贴壁 在不同的剪切率下的流室或彼此之间。我们会 通过以下方法确定血管壁成分在粘连中所起的作用 在腔体表面涂上各种细胞黏附蛋白， 通过利用来自不同部位的培养内皮细胞 通过循环和使用抗膜的单抗 阻断某些膜的蛋白质和合成肽 感受器。需要研究的一个具体的新假设是 VWF蛋白大分子多聚体的产生 局部由激活或受损的内皮细胞，起着至关重要的作用 在调节镰状红细胞/内皮细胞黏附中的作用。RBC将成为 从AS和正常人中密度分离SS组分和所有RBC 控制。要研究的变量将包括pH值的影响， 渗透压、切应力暴露、壁面切变率、钙和 钾含量与药物修饰的血红蛋白或改变的红细胞 膜。通过使用AS和AA RBC和实验 手法(增加红细胞钙，脱水，增加 膜结合血红蛋白)，我们计划生产 镰刀状红细胞。模型以及正常细胞和镰状细胞将 被流变学研究。我们的技术将涉及到剪掉 粘度计(大米粘度计或流变学流体)中的细胞 流变仪)。他们对剪切的反应将使用以下方法进行评估 生物物理和生化分析。

The objectives of the proposed work are to utilize modern rheological techniques combined with biochemical analyses to understand the biophysical basis of the pathophysiology of sickle cell anemia. This knowledge will then provide a basis for the rational development of new clinical treatments. Specifically, two major classes of problems are to be investigated: (1) red cell- endothelial cell interactions under controlled flow conditions using epi-fluorescent video microscopy and image analysis techniques; and (2) effect of shear stress on red cell membrane permeability and the sickle hemoglobin polymerization rate and final gel material properties. Structive interactions with endothelial cells leading to lack of tissue oxygenation and vaso- occlusion due to gel formation are two possible problems that could contribute to a clinical crisis. The techniques proposed will examine these areas. The investigations require very specialized equipment--some of which is only available in our laboratories. Employing videomicroscopy of red cells under controlled flow conditions, we can discriminate whether cells adhere to the wall of a flow chamber or each other at different shear rates. We will determine the role vessel wall composition plays in adherence by coating the chamber surface with various cytoadhesive proteins, by utilizing cultured endothelial cells from various parts of the circulation and by using monoclonal antibodies to membrane proteins and synthetic peptides which block certain membrane receptors. A specific novel hypothesis to be investigated is that the large molecular weight multimers of vWF protein, generated locally by activated or damaged endothelium, play a crucial role in modulating sickle RBC/endothelial cell adhesion. RBCs will be density separated SS fractions and all RBC from AS and normal controls. Variables to study will include the effect of pH, osmolarity, shear stress exposure, wall shear rate, calcium and potassium content and drug modified hemoglobin or altered RBC membranes. By using AS and AA RBC and experimental manipulations (increased red cell calcium, dehydration, increased membrane bound hemoglobin), we plan to produce models of the sickle red cell. The models as well as normal and sickle cells will be studied rheologically. Our technique will involve shearing the cells in viscometers (Rice Viscometer or Rheometrics Fluids Rheometer). Their response to shear will be assessed using biophysical and biochemical analyses.