实时细胞分析技术结合模式识别评价祖师麻注射液类过敏反应

Real-time cell analysis combined with pattern recognition to evaluate anaphylaxis of Zushima Injection

建立一种体外类过敏反应评价方法,快速评价不同厂家15批次祖师麻注射液的类过敏反应。采用体外嗜碱性白血病细胞株RBL-2H3细胞模型,选择Compound 48/80为阳性药,利用实时细胞分析(real-time cell analysis,RTCA)系统检测药物干预后引起的细胞指数(cell index,CI)变化,并利用甲苯胺蓝染色法观察细胞形态、骨架变化验证RBL-2H3细胞的脱颗粒情况。采用聚类方法对15批祖师麻注射液作用于RBL-2H3细胞后的CI进行分析。结果表明,Compound 48/80(20μg·mL-1)使细胞形态和细胞骨架均发生明显改变,发生明显的脱颗粒现象。CI在加药30 min内快速下降,之后下降趋势变缓,提示RTCA系统可以快速敏感地评价RBL-2H3细胞脱颗粒作用。15批祖师麻注射液对RBL-2H3细胞的CI结合聚类分析(CA),提示不同厂家的祖师麻注射液对RBL-2H3细胞影响不同,S1~S8与Compound 48/80组聚为一类,提示该样品可能潜在临床类过敏反应;S9~S15与空白对照组聚为一类,提示该样品无类过敏样反应。该研究建立了一套基于RTCA系统结合模式识别的类过敏反应体外快速评价技术,可用于中药注射剂类过敏反应的体外快速评价。

This paper was aimed to establish a new method for evaluating the anaphylactoid reaction of 15 batches of Zushima Injection from different manufacturers in vitro.Basophilic leukemia cell line RBL-2 H3 cells were cultured in vitro and Compound 48/80 was selected as positive drug.Real-time cell analysis(RTCA)system was used to detect the changes of cell index(CI)value after drug intervention.The degranulation of RBL-2 H3 cells was verified with the toluidine blue staining technology by observing the changes of cell morphology and skeleton.Clustering method was used to analyze the CI values of 15 batches of Zushima Injection on RBL-2 H3 cells.The results showed Compound 48/80(20μg·mL-1)significantly changed the cell morphology and cytoskeleton,with obvious degranulation.After adding Compound 48/80,CI value decreased rapidly within 30 minutes,then decreased slowly,suggesting that RTCA system can be used for rapid and sensitive evaluation of RBL-2 H3 cell degranulation.The results of cluster analysis showed that Zushima Injection from different manufacturers had different effects on RBL-2 H3 cells.S1-S8 and Compound 48/80 groups were grouped into one cluster,which suggesting that the sample might have potential clinical anaphylaxis.S9-S15 and the normal control group were grouped into one cluster,suggesting there was no anaphylactoid reaction in the sample.In this study,a rapid in vitro anaphylaxis evaluation technique based on RTCA system and pattern recognition method was established,which can be used for rapid in vitro evaluation of anaphylaxis for traditional Chinese medicine injection.