【作者】 查庆兵；

【导师】 何贤辉；

【作者基本信息】 暨南大学 ， 免疫调节与免疫药理， 2006， 硕士

【摘要】 目的： 利用负载人巨细胞病毒（HCMV）结构蛋白pp65衍生抗原肽NLV(pp65_495-503)的HLA-A*0201（A2-NLV）四聚体，探讨四聚体染色条件的优化，比较健康的中国青年人、老年人HCMV特异性CTL频率和表型，并分析HCMV抗原肽NLV特异性CTL细胞的功能特性和TCR Vβ的取用。 方法： 1．以HLA-A2⁺供者外周血为实验材料，在不同条件下进行A2-NLV四聚体／PE染色，然后以anti-CD3-FITC和anti-CD8-APC标记，流式细胞术分析染色结果，寻找四聚体最佳染色条件。 2．取中国健康老年人和年轻人HLA-A2⁺供者外周血，以A2-NLV四聚体／PE和anti-CD8-APC、anti-CD28-FITC或anti-CD57-FITC（表型抗体）标记，流式细胞术分析染色结果。 3．将HLA-A2／NLV四聚体染色与细胞内细胞因子或穿孔素染色相结合，以流式细胞仪直接分析NLV特异性CTL细胞内穿孔素的表达水平，或者在NLV抗原肽刺激6h后分析γ-干扰素（IFN-γ）的表达水平。更多还原

【Abstract】 AIM: To optimize tetramer staining condition using HLA-A*0201 tetramer（A2-NLV tetramer） loaded with NLV peptide (pp65_495-503 derived from structural protein pp65 of human cytomegalovirus,and to investigate the phenotypic and functional characteristics and TCR Vβ usage of HCMV-specific CTL cells in healthy young and elderly Chinese donors.METHODS:1. Peripheral blood from HLA-A2⁺ donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition.2. Whole blood from healthy HLA-A2⁺ young and elderly Chinese donors were stained with a combination of the tetramers and antibodies for CD28 and CD57 and were analyzed by three-color flow cytometry.3. Perforin expression was analyzed with flow cytometry by using A2-NLV tetramer staining together with intracellular staining without stimulation, while γ-interferon更多还原