RHEOLOGICAL STUDIES OF SICKLE CELL DISEASE

拟议工作的目标是利用现代 流变学技术结合生化分析， 了解镰刀病的病理生理学的生物物理学基础 细胞性贫血 这些知识将为我们提供一个基础， 合理开发新的临床治疗方法。 具体地说， 要研究两大类问题：（1）红细胞- 受控流动条件下的内皮细胞相互作用 使用落射荧光视频显微镜和图像分析 剪切应力对红细胞膜的影响 渗透性和镰状血红蛋白聚合率， 最终凝胶材料性质。 结构性互动， 内皮细胞导致缺乏组织氧合和血管， 由于凝胶形成的堵塞是两个可能的问题， 可能导致临床危机 所提出的技术将 检查这些地区。 调查需要非常专业的 设备--其中一些只有我们的实验室才有。 在受控流下使用红细胞的视频显微镜 条件下，我们可以区分细胞是否粘附在壁上， 或彼此在不同的剪切速率下。 我们将 确定血管壁成分在粘附中的作用， 用各种细胞粘附蛋白涂覆腔室表面， 通过利用来自不同部位的培养的内皮细胞， 循环和使用单克隆抗体膜 蛋白质和合成肽， 受体。 有待研究的一个具体的新假设是， vWF蛋白的大分子量多聚体，产生 局部激活或受损的内皮细胞，发挥至关重要的作用， 调节镰状红细胞/内皮细胞粘附。 RBC将 密度分离SS组分和AS和正常人的所有RBC 对照 研究的变量将包括pH值的影响， 渗透压，剪切应力暴露，壁剪切率，钙和 钾含量和药物修饰的血红蛋白或改变的红细胞 膜。 用AS和AA红细胞， 操作（增加红细胞钙，脱水，增加 膜结合血红蛋白），我们计划生产的模型， 镰状红细胞 该模型以及正常细胞和镰状细胞将 从流变学的角度来研究。 我们的技术将涉及剪切 粘度计（Rice Viscometer或Rheometrics Fluids）中的细胞 流变仪）。 它们对剪切力的反应将使用 生物物理和生物化学分析。

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.