艾滋病猴特异性细胞免疫的胞内细胞因子检测方法优化与应用

目的 为准确检测艾滋病猴模型特异性细胞免疫,优化、确定胞内细胞因子染色(ICS)影响因素和条件.方法 使用三种多克隆激活剂分别刺激SIV感染猴外周血单个核细胞(PBMC),确定最佳阳性刺激物和刺激时间；然后使用五种浓度SIVmac239混合肽库分别刺激SIV感染猴PBMC,体外培养,不同时间点进行细胞染色和流式检测,确定肽库的最适刺激浓度和最佳刺激时间.最后,初步应用该方法检测SIV感染猴细胞免疫水平.结果 PMA+离子霉素组合可用作本实验的阳性刺激物；2μg/mL肽库,37℃5％ CO2培养16 h,能更有效的刺激T细胞分泌TNF-α、IL-2、IFN-γ.结论 该方法的优化对艾滋病药物的临床前评价和疫苗研发等研究具有重要意义.

Improvement and application of intracellular cytokines staining assay to detect SIVmac239-specific cellular immunity in SIV-infected rhesus monkeys

Objective To improve the intracellular cytokines staining （ICS） assay and serve the detection of SIVspecific cellular immunity in SIV-infected rhesus monkeys. Methods Three polyelonal activators were used for PBMC stimulation, and the best positive stimulators and the reasonable stimulating time were tested and determined. Five concentrations of SIVmae239 mixed peptides pool were designed to stimulate PBMC from SIV-infeeted rhesus monkeys, followed by culturing in vitro and detected with flow cytometry at different times, and the optimal concentration of SIV mixed peptides pool and the best stimulating time were ascertained finally. Then, this method was used for detection of the SIV-specific cellular immunity in the monkeys. Results Combination of PMA ＋ ionomycin could be used as the best positive stimulator. Cytokines secreted from T cells were detected at the highest level by 2 p.g/mL SIVmac239 mixed peptides pool at 37°C and in 5% CO2 culturing for 16 h in vitro. Conclusions Optimization of the experimental conditions of ICS has been successfully accomplished, and it may be useful for pre-clinical evaluation of AIDS drugs and vaccine research.