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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(112%±013%),而分泌IL-4和IL-10的T淋巴细胞则无升高(分别为012%±003%和010%±003%);分泌IFN-γ的T淋巴细胞可以被进一步纯化(938±221)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。
Objective To measure and purify cytokines secreted by allogeneic peripheral blood mononuclear cells (PBMNCs), and to provide a new approach for the study of allogeneic T lymphocytes. Methods T lymphocytes secreting IFN-γ, IL-4 and IL-10 in human mixed lymphocyte reaction were quantitatively determined by a novel cytokine secretion assay (CKSA) at the single-cell level. T-cells secreting IFN- γ Magnetic purification. Results The levels of T lymphocytes secreting IFN-γ were significantly increased after allogeneic PBMNCs were stimulated (112% ± 013%), while the levels of T-lymphocytes secreting IL-4 and IL-10 were not increased % ± 003% and 010% ± 003%); IFN-γ secreting T lymphocytes can be further purified (938 ± 221) fold. Conclusions The level of T lymphocytes secreting IFN-γ after allogeneic PBMNCs were stimulated with CKSA was quantitatively determined from a single cell level. These cells could be efficiently purified.