单个CD8^+T细胞内IFN-γ含量的测定  

Detection of IFN-γ level in single CD8^+ T cell

作　　者：王志杰[1] 张华[2] 陈学良[1] 金文睿[3] 丛雅琴[1] 韩波[1] 谷燕[1] 

机构地区：[1]山东大学齐鲁医院血液科,山东济南250012 [2]山东大学生命科学学院微生物技术国家重点实验室,山东济南250100 [3]山东大学化学与化工学院分析化学教研室,山东济南250100

出　　处：《细胞与分子免疫学杂志》2005年第1期72-75,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省自然科学基金资助项目(No.Y2001C09)

摘　　要：目的: 建立一种新的单细胞内IFN -γ的检测方法毛细管电泳免疫分析激光诱导荧光法 (CEIA- LIF)。方法:选 2例重型再障(severaplasticanemia, SAA)患者及 1例正常人, 用免疫磁珠法分离纯化外周血CD8+ T细胞。将其与毛地黄皂苷反应 15min, 再与FITC 抗IFN -γ单抗 (mAb)反应20min。用CEIA -LIF法进行单个细胞的连续检测, 并用倒置显微镜及激光扫描共聚焦荧光显微镜等证实本法的可行性。结果: 纯化的CD8+ T细胞内的IFN γ, 是在不溶膜的情况下得到全部检测。2例SAA患者外周血单个CD8+ T细胞内IFN -γ的含量, 分别为 ( 151. 53±28. 92 )zmol和 ( 223. 72±45. 23)zmol, 高出正常对照 ( 47. 47±17. 97 )zmol约 3 ~4倍(P<0. 01)。结论: CEIA LIF法对单个CD8+ T细胞内IFN -γ含量的测定是可行的, 有望用于临床检测。AIM: To establish a new method capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF), to detect IFN-γ level in single CD8+ T cell. METHODS: CD8+ T cells were isolated from peripheral blood of two patients with severe aplastic anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation and then were purified by immunomagnetic microbead separation. Then purified CD8+ T cells were incubated with digitonin for 15 min followed by FITC-anti-IFN-γ mAb for 20 min. The single cell was detected continuously by CEIA-LIF. The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy. RESULTS: The IFN-γ content in purified CD8+ T cells was detected under the condition of cell-membrane integrity. The IFN-γ level in single CD8+ T cell from 2 SAA patients was (151.53±28.92)zmol and (223.72±45.23)zmol, respectively, and much higher that from normal control (47.47±17.97)zmol (P=0.001). CONCLUSION: It is feasible to quantitate IFN-γ in single CD8+ T cell by CEIA-LIF. CEIA-LIF might be useful in the clinical detection of intracellular cytokines.

关 键 词：CD8^+T细胞 Γ-干扰素 毛细管电泳免疫分析 激光诱导荧光检测 

分 类 号：R446.61[医药卫生—诊断学]