摘要
T细胞增殖反应是宿主T细胞识别病原的结果,也是宿主细胞免疫应答的重要指标之一。为了便于检测猪群在病原感染或者疫苗免疫过程中产生的细胞免疫应答,本研究应用MTT法建立了体外检测猪外周血T细胞增殖反应的研究方法。通过密度梯度离心法从外周血分离得到外周血单个核细胞(PBMC),然后利用单核细胞和淋巴细胞不同的生长特性(贴壁与否),弃掉贴壁的单核细胞,获得外周血淋巴细胞(PBL)。外周血淋巴细胞的流式分析结果显示,分离获得的PBL中T细胞所占比例达到了80%以上。应用MTT法分析了非特异性刺激物刀豆蛋白A(ConA)的浓度和细胞培养密度对T细胞增殖的影响。结果显示,ConA的工作浓度为5μg/mL、细胞培养密度为2×106/mL时T细胞的增殖反应最强烈。本研究所建立的猪外周血T细胞增殖反应检测法可以为研究猪针对病原或疫苗的细胞免疫反应提供参考。
Recognition of MHC-peptide complexes by T lymphocytes results in robust T cell proliferation,which is regarded as one of the hallmarks of the host cell-mediated immune responses against pathogens.To conveniently detect cell-mediated immune responses of swine upon pathogen infection or vaccine immunization,we developed a method for studying peripheral blood T cell proliferation of swine via MTT assay.Firstly,peripheral blood mononuclear cells（PBMC） were collected through density gradient centrifugation,and then peripheral blood lymphocytes（PBL） were obtained by discarding adherent monocytes after co-culture overnight.Flow cytometry analysis showed that the percentage of T cells in PBL was 80%.Next,the MTT assay was used to detect effects of the concentration of ConA and cell culture density on porcine T cell proliferation.The result of MTT suggested that when 2×106/mL T cells were cultured with 5 μg/mL ConA,the highest level of T cell proliferation was achieved.The MTT assay for studying porcine T cell proliferation could facilitate assessing the T cell mediated immune response against pathogens or vaccines.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第12期35-38,共4页
China Animal Husbandry & Veterinary Medicine
基金
基金项目:2009ZX08009-152B
