Design of a high-sensitivity lipid particle method for cell separation

说明(申请人提供)：免疫细胞参与多种心血管疾病，包括动脉粥样硬化、心肌梗死后的心脏重构以及血管炎中的血管炎症。虽然可以用流式细胞术分析血管壁中的免疫细胞，但必须分离这些细胞才能用于过继转移和免疫重建实验。目前的方法不能以足够的效率、活性和纯度分离感兴趣的细胞。这是进一步研究的障碍。与动脉粥样硬化相关的免疫细胞很少(每只小鼠大约3000个)，因此很难从动脉粥样硬化病变的环境中分离出来。这里提出的工作旨在设计一种新的技术来识别、选择和分离高价值细胞靶标。动脉粥样硬化与对低密度脂蛋白(LDL)中的主要脂蛋白ApoB-100的免疫反应有关。具体地说，感兴趣的目标是针对可能调节动脉粥样硬化的自体肽--来自ApoB-100的SQEYSGSVANEANVY和TGAYSNASSTESASY--的T细胞。在动脉粥样硬化预防接种策略中使用这些多肽的成功工作激发了对这些抗原特异性T细胞的详细研究。为了完成这些细胞的分离，我建议将充满气体的脂质微泡偶联到与其中一个抗原肽结合的II类主要组织相容性复合体(MHC II)多聚体。在靶细胞上的反应性T细胞受体(TCR)与其多聚体/微泡复合体上的同源MHC-II/肽结合后，离心浮力微泡作为一种积极的选择机制来分离这些细胞。该项目涉及基于微气泡的浮力细胞分离的设计和验证。它的目的是解决目前细胞分离技术的纯度、活性和得率低的问题。这些问题是动脉粥样硬化领域取得进展的关键障碍。通过该项目开发的新技术将为未来的心血管疾病研究提供有用的平台。

DESCRIPTION (provided by applicant): Immune cells are involved in a variety of cardiovascular diseases, including atherosclerosis, cardiac remodeling following myocardial infarction, and blood vessel inflammation in vasculitis. Although immune cells contained in blood vessel walls may be analyzed using flow cytometry, these cells must be separated in order to use them for adoptive transfer and immune reconstitution experiments. Current methodologies do not separate cells of interest with sufficient efficiency, viability, and purity. This is a barrer to further research. Immune cells of interest in the context of atherosclerosis are rare (approximately 3000 per mouse aorta) and therefore difficult to separate from the milieu of an atherosclerotic lesion. The work proposed here is aimed at designing a novel technique for identifying, selecting, and isolating high- value cell targets. Atherosclerosis is associated with n immune response to ApoB-100, the main lipoprotein in low density lipoprotein (LDL). Specifically, the targets of interest are T cells specific to self-peptides-SQEYSGSVANEANVY and TGAYSNASSTESASY, from ApoB-100- that may modulate atherosclerosis. Successful work involving the use of these peptides in an atheroprotective vaccination strategy motivates detailed study of these antigen-specific T cells. To accomplish separation of these cells, I propose to couple gas-filled lipid microbubbles to class II major histocompatibility complex (MHC II) multimers binding one of the antigenic peptides. After engagement of reactive T-cell receptors (TCR) on target cells to their cognate MHC-II/peptide on the multimer/microbubble complex, centrifuged buoyant microbubbles function as a positive selection mechanism to separate these cells. This project involves the design and validation of microbubble-based buoyant cell separation. Its intention is to solve the problems of low purity, viability, and yieldof current cell separation techniques. These problems present a critical barrier to progress in the field of atherosclerosis. The new technology developed through this project will be a useful platform for future cardiovascular disease research.