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、富集和扩增，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干扰素-γ（IFN-γ）的T淋巴细胞；并对其进行磁性富集、扩增，再用酶联免疫斑点法（ELISPOT）检测其对原抗原的特异性。结杲：同种异体PBMNCs刺激后检测到分泌IFN-γ的T淋巴细胞水平较对照组明显升高[（1．12±0．13）%vs（0．23±0．07）%，P〈0．05]，进一步富集达（67．3±10．5）%，相对倍数为（93．8±22．1）倍。经过富集的细胞能够通过OKT3、抗CD28单抗和IL-2组合，在21～28d扩增〉600倍，扩增后的细胞保留与原相关异基因刺激细胞特异性反应，并具分泌IFN-γ的能力。结论：利用CKSA从单细胞水平定量测定到显著升高的由同种异体PBMNCs刺激后分泌IFN-γ的T淋巴细胞。这些细胞可以被有效地富集和扩增，并保留针对原抗原的特异反应性。
Objective： To detect,enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation. Methods： The novel cytokine secretion assay （CKSA）was applied to detect T lymphocytes secreting IFN-γ at single cell level in human mixed lymphocytes reaction. IFN-γ secreting T cells were enriched by means of magnetic sorting system and expanded with OKT3,anti-CD28mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay, Results： A sizable proportion of IFN-γ secreting T lymphocytes could be detected [（1.12±0. 13）%vs （0.23±0.07）%] and be further enriched to （67.3±10.5）%,or （93.8±22.1） fold. Tlymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-γ response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control. Conclusion： It is feasible to detect significantly increased IFN-γ secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique atsingle cell level and these cells can be efficiently enriched and expanded for further research.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2006年第1期39-44,共6页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金(30270571)
浙江省医药卫生研究基金(2004B030)
