类过敏反应体外评价模型的建立及其指标的筛选  

Establishment of in vitro evaluation model of pseudoallergy and screening of its indexes

作　　者：李伟[1] 陈梦[1,2] 黄芝瑛[2] 姜华[1] 李波[1] 

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176 [2]中山大学药学院,广州510006

出　　处：《中国新药杂志》2017年第14期1649-1660,共12页Chinese Journal of New Drugs

基　　金：国家“重大新药创制”科技重大专项资助项目(2012ZX09302001)

摘　　要：目的:以C48/80为阳性药物,比较3种细胞系(RBL-2H3,P8l5和Ku812细胞)及其指标在评价药物类过敏中的敏感性;并采用聚山梨酯80对3种细胞模型评价类过敏反应的适用性进行考察。方法:以不同浓度的C48/80分别作用于3种细胞系30和60 min,测定β-氨基糖苷酶释放率、Annexin V阳性细胞率以及IL-4和TNF-α释放量。然后,用聚山梨酯80进行模型的适用性考察,同时对不同浓度的聚山梨酯80引起的类过敏反应进行评价。结果:C48/80可引起3种细胞系的β-氨基糖苷酶释放率、Annexin V阳性细胞率、IL-4释放量和TNF-α释放量呈剂量依赖性增加;除了RBL-2H3细胞,其他2个细胞系检测到的IL-4释放量非常少,且3种细胞系检测到的TNF-a量亦非常少;Annexin V阳性细胞率的增加程度较其他3项指标明显;在相同刺激条件下,P815和Ku812细胞的β-氨基糖苷酶释放率和Annexin V阳性细胞率的突增较RBL-2H3细胞发生时间早,突增效果更明显。聚山梨酯80的结果与C48/80给药时基本吻合。此外,还发现聚山梨酯80≥0.05 mg·m L^(-1)时,即存在引起类过敏反应的风险。结论:β-氨基己糖苷酶释放率和Annexin V阳性细胞率是评价类过敏反应的2种稳定、可靠、重复性好的检测指标;IL-4释放量和TNF-α释放量较适合作为辅助检测指标;Annexin V阳性率可较敏感地、较早地提示类过敏反应的发生。P815和Ku812细胞较RBL-2H3细胞更适合作为一种早期、稳定、敏感的肥大细胞脱颗粒体外检测模型,用于致类过敏反应药物的早期筛选。Objective： To compare the sensitivity of three kinds of cell lines （RBL-2H3, P815 and Ku812 cells） and their markers in the evaluation of drug pseudoallergy and explore the applicability of the in vitro models for the evaluation of pseudoallergy. Methods： C48/80 was used as the positive drug. The cells were treated with different concentrations of C48/80 for 30 and 60 min, respectively. Cell degranulation was evaluated by detecting the release rate of 15-hexosaminidase, the ratio of Annexin V positive cells, and the release amounts of IL-4 and TNF-alpha. Then, the applicability of the models was studied though testing the reported anaphylactoid reaction drug Tween 80. At the same time, the pseudoallergy induced by different concentrations of Tween 80 was evaluated.Results： After treatment with C48/80, the release rate of [3-hexosaminidase, the ratio of Annexin V positive ceils, and the release amounts of IL-4 and TNF-alpha in three kinds of cells were all significantly increased in a dose- dependent manner. However, except for the RBL-2H3 cells, the amount of IL-4 released in the other two cell lines was very small, and the amount of TNF-a detected in all three kinds of cell lines was very small. The rate of Annexin V positive cells increased significantly compared with the other three indicators. In the same stimulating conditions, the percentage of Annexin V positive cells and the release rates of [3-hexosaminidase in P815 and Ku812 cells increased significantly earlier than that of RBL-2H3 cells, and the burst effect was more obvious. The results of Tween 80 administration were basically identical with that of C48/80 administration. In addition, the study also found that when the concentration of Tween 80 was greater than O. 05 mg- mL-I , there was a risk of causing allergic reaction. Conclusion： [3-hexosaminidase release rate and AnnexinV positive cell rate are two stable, reliable and reproducible indexes for the evaluation of pseudoallergy. IL-4 and TNF-alpha release are more suitable as auxilia

关 键 词：类过敏反应 RBL-2H3细胞 P8l5细胞 Ku812细胞 体外 

分 类 号：R28[医药卫生—中药学] R932[医药卫生—中医学]