中文摘要：

目的建立流式细胞术检测小鼠细胞内细胞因子的方法。方法取5只BALB/c小鼠建立哮喘模型,4周后处死,制备脾脏单个核淋巴细胞悬液,分别按A、B、C3个刺激方案培养。A方案组：用25ng/mlPMA、1μg/mlIonomy-cin以及10μg/mlBFA刺激4h;B方案组用50ng/mlPMA、1μg/mlIonomycin以及10μg/mlBFA刺激4h;C方案组用3μg/mlConA刺激2d,加入10μg/mlanti-CD3、2μg/mlanti-CD28和10μg/mlBFA后继续刺激5h。培养后各组细胞悬液根据检测目的分成若干检测管,结合固定破膜处理技术,进行细胞表面抗原CD4及细胞内因子IL-4、IL-5、IL-10和IFN-γ染色,用流式细胞仪分析,比较不同刺激条件下各种细胞内因子的表达情况。实验设未刺激对照、激活对照、胞内染色对照及同型对照。结果 A方案组中5只鼠的细胞内因子表达率IL-4为（5.96±1.199）%,IFN-γ为（7.98±3.939）%,IL-10为（4.78±2.366）%,IL-5表达率均低于1%;B方案组中5只鼠的细胞内因子表达率IL-4为（5.76±1.108）%,IFN-γ为（7.44±2.269）%,IL-10为（4.50±1.560）%,IL-5表达率均低于1%;C方案组5只鼠IL-10、IL-5表达率分别为（3.14±0.288）%和（6.82±1.996）%。结论 25ng/mlPMA、1μg/mlIonomycin及10μg/mlBFA刺激4h的方案较适合小鼠淋巴细胞IL-4、IFN-γ的检测;3μg/mlConA刺激2d,加入10μg/mlanti-CD3、2μg/mlanti-CD28和10μg/mlBFA继续刺激5h的方案适合IL-5、IL-10的检测,并且均是从单个核细胞水平对细胞内因子进行检测。

英文摘要：

Objective To develop a method of measuring intracellular cytokines in mice using flow cytometry.MethodsAsthma was induced in 5 BALB/c mice that sacrificed four weeks later,when their spleens were aseptically collected.Splenic cells were isolated and divided into three groups for culturing：Group A consisted of cells cultured with 25 ng/ml PMA and 1 μg/ml ionomycin in the presence of 10 μg/ml BFA for 4 h,Group B consisted of cells cultured with 50 ng/ml PMA and 1 μg/ml ionomycin in the presence of 10 μg/ml BFA for 4 h,and Group C consisted of cells cultured with 3 μg/ml ConA for 2 d and then 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 in the presence of 10 μg/ml BFA for 5 h.Suspensions of each group of cells were divided among several tubes for different uses.Cells were stained for surface CD4 and then the intracellular cytokines IL-4,IL-5,IL-10,and IFN-γ after combined fixation and permeabilization.Cytokines were assessed with flow cytometry.Relevant controls were set up in the experiment.Results The rate of intracellular cytokine expression for each Group was as follows.Group A：（5.96±1.199）% IL-4,（7.98±3.939）% IFN-γ,（4.78±2.366）% IL-10,and less than 1% IL-5.Group B：（5.76±1.108）% IL-4,（7.44±2.269）% IFN-γ,（4.50±1.560）% IL-10,and less than 1% IL-5.Group C：（3.14±0.288）% IL-10,and （6.82±1.996）% IL-5.Conclusion IL-4 and IFN-γ were detected in mice lymphocytes with 25 ng/ml PMA and 1 μg/ml ionomycin in the presence of 10 μg/ml BFA for 4 h.IL-5 and IL-10 were detected with 3 μg/ml ConA for 2 d,10 μg/ml anti-CD3,and 2 μg/ml anti-CD28 in the presence of 10 μg/ml BFA for 5 h.Detection was accomplished at the level of mononuclear cells.