基于细胞因子分泌对人混合淋巴细胞反应中的T淋巴细胞进行测定和纯化

摘要

目的:对同种异体外周血单个核细胞(PBMNCs)刺激后分泌细胞因子的T 淋巴细胞进行测定和纯化,为研究介导同种异体反应的T淋巴细胞提供新的途径.方法: 利用新颖的细胞因子分泌检测方法(CKSA)从单细胞水平定量测定人混合淋巴细胞反应中分泌IFN-γ,IL-4 和IL-10的T 淋巴细胞; 对分泌IFN-γ的T细胞进行磁性纯化.结果: 同种异体PBMNCs刺激后检测到分泌IFN-γ的T淋巴细胞水平明显升高(1.12%±0.13%),而分泌IL-4 和IL-10的T淋巴细胞则无升高(分别为0.12%±0.03%和0.10%±0.03%);分泌IFN-γ的T淋巴细胞可以被进一步纯化(93.8±22.1)倍.结论: 利用CKSA从单细胞水平定量测定同种异体PBMNCs刺激后分泌IFN-γ的T淋巴细胞水平显著升高,这些细胞可以被有效地纯化.%AIM: Detection and enrichment of T lymphocytes after allogeneic PBMNC stimulation according to secreted cytokine were performed in order to explore a new approach for studying allogeneic reactive T lymphocytes. METHODS: The novel cytokine secretion assay (CKSA) was applied to detect T lymphocyte secreting IFN-γ, IL-4 and IL-10 at single cell level in human mixed lymphocyte reaction. IFN-γ secreting T cells were enriched by means of magnetic sorting system. RESULTS: Allogeneic PBMNC stimulation didn't alter the proportion of IL-4 and IL-10 secreting T lymphocytes (which were 0.12%±0.03% and 0.10%±0.03%, respectively), but increased proportion of IFN-γ secreting T lymphocytes (1.12%±0.13%). These IFN-γ- secreting T lymphocytes could be further enriched to 67.3%±10.5% [(93.8±22.1) fold]. CONCLUSION: It is feasible to detect significantly increased IFN-γ-secreting T cells after allogeneic PBMNC stimulation based on the novel CKSA technique, and these cells could be efficiently enriched for further use.