ACTIVATION AND SENESCENCE OF HELPER T LYMPHOCYTES

该提案的目标是开发和应用一种创新的 技术来评估人类T细胞的有丝分裂历史， 细胞基础 拟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）， 流式细胞仪常规免疫细胞计数，以确定 端粒末端限制性片段（TRF）长度，这是一个直接的 反映T细胞有丝分裂史和增殖潜力。 到 为实现这一研究目标，PI计划：1）优化设计 发夹分子信标，用于定量 端粒TRF; 2）完善固定和杂交方案， 悬浮液中PBMC样品的定量评估; 3）验证TRF 使用基于信标强度分类的细胞的长度; 4）比较不同的 信标缀合物，以优化与可获得的市售 用于细胞表面标志物的抗体;和5）关联FACS定量 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， 将对来自复发性骨髓瘤患者的PBMC样品进行分析。 妊娠丢失（作为疾病模型）和HIV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.