ACTIVATION AND SENESCENCE OF HELPER T LYMPHOCYTES

这项提案的目标是开发和应用一种创新的 一种评估人T细胞有丝分裂历史的技术 单元格基础。提议的新方法将利用这些属性 一种新的核酸探针(称为分子信标)的上下文 用流式细胞仪测定常规多色免疫细胞计数法 端粒末端限制片段(TRF)长度，这是一个直接的 反映T细胞有丝分裂史和增殖潜能。至 为了实现这一研究目标，PI计划：1)优化设计 一种用于定量六聚体重复序列的发夹分子信标 端粒TRF；2)完善固定和杂交方案 悬液中外周血单个核细胞定量检测；3)验证TRF 使用信标强度排序的信元长度；4)比较不同 Beacon共轭以优化与现有商业广告的兼容性 细胞表面标志物的抗体；以及5)与FACS定量相关 端粒重复序列与T细胞体外复制能力的关系。 为了证明这种新方法的研究适用性，体外 将对复发患者的PBMC样本进行分析 妊娠丢失(作为一种疾病模型)和艾滋病毒患者 感染。

The goal of this proposal is to develop and apply an innovative technique to assess the mitotic history of human T cells on a single- cell basis. The proposed new methodology will exploit the attributes of a novel nucleic acid probe (termed a molecular beacon) in the context of conventional multicolor immunocytometry by FACS to determine telomeric terminal restriction fragment (TRF) length, which is a direct reflection of T cell mitotic history and proliferative potential. To achieve this research goal, the PI plans to: 1) optimize the design of a hairpin molecular beacon to quantitate the hexameric repeats of telomeric TRF; 2) refine the fixation and hybridization protocols for quantitative assessment in PBMC samples in suspension; 3) validate TRF lengths using cells sorted on beacon intensity; 4) compare different beacon conjugates to optimize compatibility with available commercial antibodies for cell surface markers; and 5) correlate FACS quantitation of telomeric repeats with the replicative capacity of T cells in vitro. To demonstrate the research applicability of this new method, ex vivo analysis will be performed on PBMC samples from patients with Recurrent Pregnancy Loss (as a disease model) and from patients with HIV infection.