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目的建立THP-1分化巨噬细胞用于检测细胞免疫的体外方法。方法将HPV治疗性疫苗原液参考品及样品用含佛波酯(phorbol myristate acetate,PMA)的RPMI1640培养基稀释后,加入THP-1细胞,设PMA对照(只加PMA),37℃,5%CO2培养箱孵育,取上清,检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的含量,测定样品体外相对效力。按上述方法对细胞密度(2.0×105、5.0×105、10.0×105个/ml)、PMA终浓度(2.8、8.3、25、75、225 ng/ml)和培养时间(2、3、4 d)进行优化,确定最佳检测条件。用HPV预防性疫苗原液及ELISPOT法验证该方法的有效性。结果优化的最佳检测条件为:细胞密度5.0×105个/ml,PMA终浓度25 ng/ml,培养时间3 d。验证结果表明该方法有效可行。结论建立了THP-1分化巨噬细胞用于检测细胞免疫的体外方法,为检测治疗性疫苗的细胞免疫提供了一个体外检测平台。
Objective To establish an in vitro method for detecting cellular immunity using THP-1 differentiated macrophages. Methods The reference materials for HPV therapeutic vaccine and samples were diluted with RPMI1640 medium containing phorbol myristate acetate (PMA) and then added to THP-1 cells. PMA control (PMA only), 5% CO2 incubator, the supernatant was taken and the content of tumor necrosis factor-α (TNF-α) was measured to determine the relative in vitro potency of the sample. Cell density (2.0 × 105, 5.0 × 105, 10.0 × 105 cells / ml), PMA final concentration (2.8,8.3,25,75,225 ng / ml) and culture time (2,3,4 d ) To determine the best test conditions. The effectiveness of this method was validated using the HPV prophylactic vaccine stock and the ELISPOT assay. The optimized results were as follows: the cell density was 5.0 × 105 cells / ml, the final concentration of PMA was 25 ng / ml and the culture time was 3 days. The verification results show that this method is effective and feasible. Conclusion An in vitro method for detecting cellular immunity using THP-1 differentiated macrophages was established and an in vitro detection platform was provided for detecting cellular immunity of therapeutic vaccines.