流式细胞术检测细胞内细胞因子方法的建立

Determination of Intracellular Cytokines by Flow - Cytometry

目的建立流式细胞术检测细胞内细胞因子的方法。方法运用荧光标记的抗细胞表面抗原单抗及抗细胞因子单抗，结合固定、破膜处理技术，检测产生ＩＦＮ－γ、ＩＬ－４的ＣＤ４＋、ＣＤ8＋细胞以及产生IＬ－１０的ＣＤ３＋细胞。同时以相应同种型ＩｇＧ染色细胞作为阴性对照，以确定细胞团于染色的特异性。结果产生ＩＦＮ－γ的ＣＤ４＋及ＣＤ８＋细胞明显多于产生ＩＬ－４的细胞，并且产生ＩＦＮ－γ的ＣＤ８＋细胞百分比较ＣＤ４＋细胞相对增多；同时产生ＩＦＮ－γ／ＩＬ－４的ＣＤ４＋、ＣＤ８＋细胞以及产生ＩＬ－１０的ＣＤ３＋细胞都较少。结论该方法可从单个细胞水平直接检测全血标本中Ｔ细胞亚群和单核细胞等细胞因子的分泌。

Objective To develop the technique for measuring intracellular cytokines by3 - colour flow-cytometry. Methods Using directly labeled anticytokine antibodies withthe combination of a fixation and permeabilization treatments, we have examined IFN-rand IL- 4 production within the CD4+ and CDS+ lymphocytes from 5 normal donors. Theoptimum stimulant dose and culture time were explored. Results The results showed thatthere was a greater proportion of CDS+ cells than CD4+ cells producing IFN-rγ and therewere few CD4 or CD8 cells producing both IFN - r and IL - 4. The proportion of CD3+ cellsproducing IL-- 10 was very few. Conclusion This method can allow the determination ofcytokine production as well as the phenotype of the producer cells at the level of individualcells in whole blood sample.