3种细胞用于建立体外肥大细胞脱颗粒模型的比较研究及应用

Establishment of In-vitro Model of Mast Cell Degranulation with Three Kinds of Cell Lines and Its Application

【目的】比较3种肥大细胞(Ku812、RBL-2H3和P8l5细胞)激活后脱颗粒反应的差异,寻找适宜的肥大细胞脱颗粒体外检测模型,并利用该模型检测几种常见的中药注射剂(TCMI)以探讨肥大细胞体外研究过敏及类过敏反应的可行性。【方法】以10μg/mL C48/80激活细胞0～60 min,采用液质联用法(LC-MS)检测组胺释放量,比色法检测氨基糖苷酶释放率,中性红染色法进行形态学观察。【结果】C48/80刺激P815、RBL-2H3、Ku812细胞0～60 min后组胺释放率、氨基糖苷酶释放率和细胞脱颗粒率均有明显增加,且在相同刺激条件下,P815细胞活化后脱颗粒现象出现更早、程度更高(P<0.05)。故选择P815细胞对数种TCMI的致敏性进行体外检测,正常P815细胞出现较少脱颗粒现象,而清开灵、血塞通、双黄连、肿节风、香丹等注射液能显著引起细胞脱颗粒释放组胺。【结论】相对于RBL-2H3和Ku812细胞,P815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,其对几种常用TCMI的检测结果与临床报道有一定的相关性,P815细胞具有评价TCMI致过敏及类过敏反应的潜在应用价值。

Objective To investigate the difference between C48/80-induced degranulation in Ku812,RBL-2H3,and P815 cells,and to find an early,stable and sensitive in-vitro mast cell degranulation model for the detection of allergic and anaphylactoid reaction induced by several traditional Chinese medicinal injections（TCMI）.Methods Cells were activated with 10 μg/mL of C48/80 for 0-60 min.Cell degranulation was evaluated by detecting the release of histamine with liquid chromatography-mass chromatography（LC-MS）,examining hexosaminidase with colorimetric assays,and observing the morphological changes with neutral red staining.Results After treatment with C48/80,the histamine release rate,hexosaminidase release rate and cell degranulation rate were markedly increased in Ku812,RBL-2H3 and P815 cells（P0.05）,and the degranulation occurred earlier and was more obvious in the activated P815 cells（P0.05）.And then the in-vitro detection of the sensitization of several TCMI was performed on P815 cells.The results showed that Qingkailing Injection,Xuesaitong Injection,Shuanghuanglian Injection,Zhonjiefeng Injection and Xiangdan Injection induced obvious release of histamine in P815 cells.Conclusion Compared with Ku812 and RBL-2H3 cells,P815 cells are suitable for the early,stable and sensitive in-vitro mast cell degranulation model,and the detection results of allergic anaphylactoid reaction induced by several common TCMI show certain accordance with the clinical reports.P815 cells can be expected to evaluate the allergic and anaphylactoid reaction caused by TCMI.