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

一种高灵敏度脂质颗粒细胞分离方法的设计

• 批准号: 8784108
• 负责人: Xiaoyu Shi
• 金额: $ 3.63万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784108
• 关键词: Address; Adoptive Transfer; Antibodies; Antigens; Aorta; Apolipoprotein E; Arterial Fatty Streak; Atherosclerosis; Avidity; B-Lymphocytes; Binding; Blood Vessels; CD19 Antigens; CD19 gene; Cardiac; Cardiovascular Diseases; Cell Separation; Cell model; Cell surface; Cells; Centrifugation; Complex; Coupling; Disease Progression; Effectiveness; Engineering; Epitopes; Flow Cytometry; Fluorescence Microscopy; Future; Gases; Goals; Histocompatibility Antigens Class II; Imagery; Immune; Immune response; Immunophenotyping; Inflammation; Intention; Link; Lipids; Lipoproteins; Low-Density Lipoproteins; Major Histocompatibility Complex; Mechanics; Methodology; Methods; Microbubbles; Modeling; Monoclonal Antibodies; Mus; Myocardial Infarction; PTPRC gene; Peptides; Population; Preparation; Problem Solving; Production; Protocols documentation; Research; Science; Simulate; Splenocyte; Staining method; Stains; T-Cell Receptor; T-Lymphocyte; TCR Activation; Techniques; Technology; Testing; Vaccinated; Validation; Vasculitis; Work; apolipoprotein B-100; atheroprotective; base; design; insight; interest; mRNA Expression; new technology; novel; particle; public health relevance; receptor; reconstitution; research study; vaccination strategy

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.

描述（由申请人提供）：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塑和血管炎中的血管炎症。虽然可以使用流式细胞术分析血管壁中含有的免疫细胞，但必须分离这些细胞，以便将它们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、活力和纯度分离感兴趣的细胞。这是进一步研究的障碍。在动脉粥样硬化的背景下，感兴趣的免疫细胞是罕见的（每个小鼠主动脉约3000个），因此难以从动脉粥样硬化病变的环境中分离。这里提出的工作旨在设计一种新的技术，用于识别，选择和分离高价值的细胞目标。动脉粥样硬化与对低密度脂蛋白（LDL）中主要脂蛋白ApoB-100的免疫应答有关。具体来说，目标是对自身肽特异的T细胞-来自ApoB-100的SQEYSGSVANEANVY和TGAYSNASSTESASY-可以调节动脉粥样硬化。涉及在动脉粥样硬化保护性疫苗接种策略中使用这些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。为了实现这些细胞的分离，我建议将充气脂质微泡偶联到结合抗原肽之一的II类主要组织相容性复合物（MHC II）多聚体。在靶细胞上的反应性T细胞受体（TCR）与多聚体/微泡复合物上的其同源MHC-II/肽接合后，离心的浮力微泡用作阳性选择机制以分离这些细胞。该项目涉及基于微泡的浮力细胞分离的设计和验证。其目的是解决现有细胞分离技术存在的纯度低、活率低、得率低等问题。这些问题是动脉粥样硬化领域进展的关键障碍。通过该项目开发的新技术将成为未来心血管疾病研究的有用平台。