MOLECULAR PHENOTYPING BY CYTOMETRY--CYTOKINE EXPRESSION

细胞因子表达的分子表型分析

• 批准号: 2390783
• 负责人: CARLETON C STEWART
• 金额: $ 9.46万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-23 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390783
• 关键词: cell population study; cell sorting; cytokine receptors; flow cytometry; fluorescent in situ hybridization; gene expression; hematopoiesis; hematopoietic stem cells; human tissue; immunologic assay /test; method development; phenotype; polymerase chain reaction; receptor expression

DESCRIPTION: The purpose of the proposed research is to develop methods to simultaneously immunophenotype and molecular phenotype cells to evaluate cytokine receptor and ligand expression. Hematopoietic cells are derived from a small pool of progenitor cells and differentiate from multipotential stem cells into committed progenitor cells that expand to become functional end cells. Antibodies have been produced to some of the proteins on the membrane of hematopoietic cells but not all of them. Since a unique repertoire of proteins is displayed for each subset along the differentiation pathway, investigators seek to be able to separate and identify specific cell populations using flow cytometry. The investigators hypothesize that hematopoietic cell subsets express the unique repertoire of cytokine receptors whose engagement by ligands regulates both proliferation and differentiation of these cells. To understand the functional aspects of this differentiation process it is proposed to develop the procedures necessary to both immunophenotype and molecular phenotype. Molecular phenotyping is a term applied to describe the process of determining specific nucleic acids sequences inside a cell. Both of these methods are necessary because the specific antibodies to identify certain cell types that are available, yet few are available for measuring cytokine ligands and only some ligand receptors. Molecular phenotyping by in situ PCR combined with immunophenotyping is not yet completely reduced to practice, therefore, some development work is also necessary. The detection of nucleic acid sequences will be performed by fluorescence in situ hybridization in suspension as well as by in situ PCR. The proposal emphasizes the use of flow cytometry for detection, however, microscopic imaging will be used as well. The experiments are designed to provide information that will help understand normal hematopoiesis. The development of a reliable method for molecular phenotyping by flow cytometry will have an enormous impact on basic, translational and clinical research. There are three goals to this study. The first is to develop a reliable method for molecular phenotyping by flow cytometry that can be combined with immunophenotyping. The second is to apply this technology to define the specific cytokine receptor expressed by subsets of hematopoietic progenitor cells. The third goal is to define the ligands produced by subsets of T-cells and monocytes. The methods and results will open a new approach to the interrogation of cellular function.

描述：拟议研究的目的是开发方法 同时对细胞进行免疫表型和分子表型分析， 评价细胞因子受体和配体表达。造血细胞 来源于一小群祖细胞， 多能干细胞转化为定向祖细胞， 成为功能性的终末细胞。已经产生了一些抗体， 造血细胞膜上的蛋白质，但不是全部。 由于蛋白质的一个独特的剧目显示为每个子集沿着 分化途径，研究人员试图能够分离 并使用流式细胞术鉴定特定的细胞群。的 研究者假设造血细胞亚群表达 独特的细胞因子受体库， 调节这些细胞的增殖和分化。 到 了解这个分化过程的功能方面， 建议制定必要的程序， 分子表型分子表型是一个用来描述 确定特定核酸序列的过程， cell.这两种方法都是必要的，因为特异性抗体 以确定可用的某些细胞类型，但可用的细胞很少 用于测量细胞因子配体和仅一些配体受体。 分子 通过原位PCR结合免疫表型分析的表型分析还没有 完全减少到实践中，因此，一些开发工作也 必要核酸序列的检测将通过 悬浮液荧光原位杂交和原位杂交 PCR法 该提案强调使用流式细胞术进行检测， 然而，也将使用显微成像。了实验 旨在提供有助于了解正常 造血建立了一种可靠的分子生物学方法 通过流式细胞术进行表型分型将对基础， 转化和临床研究。 这项研究有三个目标。第一，制定可靠的 通过流式细胞术进行分子表型分型的方法 免疫表型分析二是应用这项技术来定义 造血细胞亚群表达的特异性细胞因子受体 祖细胞第三个目标是定义由以下物质产生的配体： T细胞和单核细胞的亚群。这些方法和结果将为我们提供一个新的 询问细胞功能的方法。