VISCOELASTICITY OF WHITE BLOOD CELLS

在过去的一年里，我们的研究集中在粘附性的基本机制上。 单个分子键的水平。我们把我们的努力集中在 非刺激性和刺激性中性粒细胞的黏附和脱落 从人造表面、内皮细胞和其他中性粒细胞。 我们成功地培养了两种人脐静脉内皮细胞 (HUVECs)和人微血管内皮细胞(HMVECs)在大固体上 和多孔微载体，我们正在研究抗体之间的相互作用 包覆内皮细胞的珠子。通过使用抗体包被的珠子 我们可以指定与中性粒细胞相互作用的配体，而不是中性粒细胞 内皮细胞表面的受体。我们使用的是 轻轻按下乳胶珠的正压力，乳胶珠可以在 吸管就像气缸里的活塞，靠在内皮细胞上。vbl.使用 这种微管吸力技术，我们计划测量互动 包被E-选择素抗体的珠子与 血管内皮细胞跟随E-选择素的上调 刺激内皮细胞。在我们的初步实验中，我们有 观察到粘连频率逐渐增加(对照2-3倍) 前4小时和显著增加(15-20倍对照)的频率 血管内皮细胞刺激后4-5小时内的黏附 炎症因子TNFa或IL-1a。我们计划使用这项技术来 细胞黏附受体表达的时程特征 HMVECs表面的实时监测。一旦确定了特征，这种技术将 提供一种简单但有效的方法来量化粘连事件 体外培养的细胞上的特异性受体和配体。 我们第一次测量了，我们相信， 中性粒细胞上的受体和配体(在这种情况下是抗体) 受体与另一个表面结合。到目前为止，我们只使用了乳胶珠。 包被L-选择素的抗体，这是一种重要的受体 介导中性粒细胞滚动，或膜蛋白CD45，一种普遍存在的 所有白细胞上都有受体。受体，L-选择素或 CD45在静息的中性粒细胞表面基本表达。这个 中性粒细胞被保存在一个微吸管中，以便它可以自由滑动和 像气缸中的活塞一样来回摆动，因为 油层中的振荡压力。珠子，放在另一个吸管里， 位于包含振荡吸管的吸管的入口处 中性粒细胞。中性粒细胞在设定的时间内反复接触珠子， 通常S约为0.1%，而设定的接触面积约为1% M2。偶尔，在不到20%的时间里，细胞和珠子会粘在一起。 这种粘着的频率取决于接通率和 接触区域的受体和配体。受体的数量是 根据公布的中性粒细胞总数的值进行估计 表面和我们测量的接触面积。中的配基数量 接触面积是通过注意附着频率来确定的 随着粘接事件数量的增加而稳步下降。我们相信 这种减少是由于从细胞中提取受体引起的， 从而阻止这些特定的配体进一步与 细胞膜上的受体。从对下降速度的分析 粘附力事件作为接触次数的函数，我们是 能够计算出存在于接触区域中的配体的数量为 只要我们假设这些抗原-抗体反应的失败率 与实验的开启速度和时间相比可以忽略不计。 (抗原-抗体反应具有非常高的亲和力，这意味着 与开工率相比，折扣率非常慢。)典型配体 从我们的结合实验中计算出的密度约为 每平方微米有几百个抗体。的估计值 受体密度也是同一数量级。因此，对于附着频率 大约每0.5个S就有一个受体-抗体键形成， 200个受体和200个配体在接触区，转发率 常数为：KF=[2受体-配体键/S]/[200受体200配体] =5 10-5/S。这些实验证明了实际 首次测量受体和受体的结合率 当受体和配体都在(或在)一个表面时的配体。目标是 为了将这项表征表面结合的新技术扩展到 失败率显著的情况和涉及细胞-细胞的情况 黏附，尤其是白细胞与内皮细胞的黏附。另一个目标 是使用蒙特卡罗模拟来模拟这一过程 膜上的受体。

Our studies this past year have focused on basic mechanisms of adhesion at the level of individual molecular bonds. We have focused our efforts on the adhesion and detachment of the unstimulated and stimulated neutrophil from artificial surfaces and from endothelial cells and other neutrophils. We have successfully cultured both human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) on large solid and porous microcarriers and we are studying the interaction of antibody coated beads with the endothelial cells. By using antibody-coated beads rather than neutrophils, we can specify the ligands that interact with the receptors on the surface of the endothelial cells. We are using a positive pressure to gently press a latex bead, which slides freely in a pipette like a piston in a cylinder, against the endothelial cell. Using this micropipet suction technique we plan to measure the interactive forces between beads coated with an antibody to E-Selectin and endothelial cells following the up-regulation of E-selectin caused by the stimulation of endothelial cells. In our preliminary experiments, we have observed a gradual increase (2-3x control) in adhesion frequency in the first 4 hours and a marked increase (15-20x control) in the frequency of adhesion between 4 and 5 hours after stimulation of endothelial cells by the inflammation factor TNFa or IL-1a. We plan to use this technique to characterize the time course of adhesion receptor expression on the surface of HMVECs in real time. Once characterized, this technique will provide a simple but effective way to quantify the adhesion events between specific receptors and ligands on cells in vitro. We have measured, we believe for the first time, the on-rate between receptors on neutrophils and ligands (in this case antibodies) to these receptors bound to another surface. To date we have used only latex beads coated with antibodies either to L-selectin, an important receptor that mediates neutrophil rolling, or the membrane protein CD45, a ubiquitous receptor present on all white cells. The receptors, either L-selectin or CD45, are basely expressed on the surface of a resting neutrophil. The neutrophil is held in one micropipette so that it slides freely and oscillates back and forth like a piston in a cylinder because of an oscillating pressure in a reservoir. The bead, held in another pipette, is positioned at the entrance to the pipette that contains the oscillating neutrophil. The neutrophil repeatedly contacts the bead for a set time, usually about 0.1 s, and for a set contact area that is on the order of 1 m2. Occasionally, less than 20% of the time, the cell and bead adhere. This frequency of adhesion depends on the on-rate and on the number of receptors and ligands in the contact area. The number of receptors are estimated from published values of the total number on the neutrophil surface and our measurement of the contact area. The number of ligands in the contact area are determined by noting that the adhesion frequency steadily decreases as the number of adhesive events increases. We believe this decrease is caused by the extraction of receptors from the cell, which then block these particular ligands from further binding with a receptor in the cell membrane. From an analysis of the rate of decrease of adhesion events plotted as function of the number of contacts, we are able to calculate the number of ligands present in the contact area as long as we assume that the off-rate for these antigen-antibody reactions is negligible compared to the on-rate and the time of the experiment. (Antigen-antibody reactions have very high affinities, which imply that the off-rates are very slow compared to the on-rates.) Typical ligand densities calculated from our binding experiments are on the order of a few hundred antibodies per square micron. Estimated values for the receptor density are of the same order. Thus, for an adhesion frequency of about one receptor-antibody bond formed about every 0.5 s and for, say, 200 receptors and 200 ligands in the contact area, the forward rate constant is: kf = [2 receptor-ligand bonds/s]/[200 receptors 200 ligands] = 5 10-5/s. These experiments demonstrate the feasibility of actually measuring, for the first time, the rate of binding of receptors and ligands when both receptor and ligand are at (or in) a surface. A goal is to extend this new technique for characterizing surface binding to cases where the off-rate is significant and to cases involving cell-cell adhesion, especially, white cell-endothelial cell adhesion. Another goal is to model this process using Monte-Carlo simulations of the movement of receptors in a membrane.