Morvan P Y, Picot C, Dejour R, Gillot E, Genetet B, Genetet N
Groupe Universitaire de Recherche en Immunologie Fondamentale et Appliquée, Université de Rennes 1, France.
Eur Cytokine Netw. 1994 Sep-Oct;5(5):469-80.
We present an original method for in situ hybridization (ISH) using non isotopic probes and flow cytometry analysis that permits rapid detection of lymphokine transcripts at single cell level in an in vitro activated human Jurkat T cell line and in peripheral blood T cell subsets. After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualised using FITC-conjugated anti-DIG antibody, and the resultant signal was analysed by flow cytometry. IL-2 mRNA was first detected in activated Jurkat T cells. Addition of cycloheximide 4 hours after the beginning of stimulation increased both the frequency of labelled cells and the amount of mRNA per cell, as determined by the mean fluorescence intensity. The specificity and sensitivity of IL-2 mRNA detection were tested by comparison with Northern blot analysis and in situ hybridization (ISH) with immuno-cytochemical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as early as 3 hours after in vitro stimulation with PMA and ionomycin. The frequency of positive cells and the amount of mRNA per cell peaked at 6-8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing cells reached 30-40% and 15-20%, respectively. The two lymphokines were expressed in both CD4+ and CD8+ T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4+ (60%) than in CD8+ T cells (25%), whereas IFN-gamma were preferentially transcribed by CD8+ T cells (40%). The results obtained by this method were in accordance with the data obtained by Northern blot analysis, with cellular protein content estimated by immuno-fluorescence staining, and with IL-2 titration by bioassay. We compared the performance of this method with ISH using radioactive probes.
我们提出了一种使用非同位素探针和流式细胞术分析的原位杂交（ISH）原始方法，该方法能够在体外激活的人Jurkat T细胞系和外周血T细胞亚群中，在单细胞水平快速检测淋巴因子转录本。在用佛波酯（PMA）和离子霉素或刀豆蛋白A（ConA）刺激后，细胞被固定，并与地高辛（DIG）标记的针对白细胞介素-2（IL-2）和γ干扰素（IFN-γ）的RNA反义或正义探针杂交。使用异硫氰酸荧光素（FITC）偶联的抗DIG抗体观察单个细胞中细胞因子基因表达水平，并通过流式细胞术分析产生的信号。IL-2 mRNA首先在激活的Jurkat T细胞中被检测到。刺激开始4小时后添加放线菌酮，增加了标记细胞的频率以及每个细胞的mRNA量，这通过平均荧光强度来确定。通过与Northern印迹分析以及免疫细胞化学染色的原位杂交（ISH）比较，测试了IL-2 mRNA检测的特异性和敏感性。在用PMA和离子霉素体外刺激后3小时，外周血单个核细胞（PBMC）中即可检测到IL-2和IFN-γ mRNA。阳性细胞频率和每个细胞的mRNA量在6 - 8小时达到峰值，此时含IL-2和IFN-γ mRNA的细胞百分比分别达到30 - 40%和15 - 20%。这两种淋巴因子在CD4 +和CD8 + T细胞中均有表达，但CD4 +（60%）中表达IL-2的细胞频率和每个细胞的IL-2 mRNA量高于CD8 + T细胞（25%），而IFN-γ优先由CD8 + T细胞转录（40%）。通过该方法获得的结果与通过Northern印迹分析获得的数据、通过免疫荧光染色估计的细胞蛋白质含量以及通过生物测定法进行的IL-2滴定结果一致。我们将该方法的性能与使用放射性探针的ISH进行了比较。
