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类过敏反应是药物注射剂最常见的不良反应,但现有检测方法尚存在诸多不足。该研究采用荧光标记和高内涵筛选技术建立了一种类过敏反应体外检测方法,应用RBL-2H3细胞脱颗粒模型,以细胞膜特异荧光染料FM4-64标记细胞脱颗粒囊泡回收来确定阳性细胞数,以细胞核特异荧光染料Hochest 3334标记细胞核来确定细胞总数,从而计算药物刺激后的细胞脱颗粒率。为了考察高内涵检测方法可靠性,首先采用阳性药物Compound 48/80验证了高内涵检测方法与传统过敏介质β-氨基己糖苷酶释放和小鼠耳廓皮肤蓝染检测方法间一致性,结果显示高内涵检测方法与传统检测方法高度一致,且灵敏度高于传统检测方法;然后采用30批次临床报告过敏症状丹红注射液进一步验证高内涵检测方法的可靠性,结果提示高内涵检测结果与临床报告高度一致,而且高内涵方法一定程度上能够克服注射液自身颜色干扰问题。可见,类过敏反应高内涵检测方法是一种灵敏、可靠、简便且可实现高通量筛选的新方法,适用于药物注射剂类过敏反应评价,能够为临床安全用药提供指导。
Anaphylaxis is the most common adverse reaction of drug injection, but there are still many deficiencies in the existing detection methods. In this study, fluorescence-labeled and high-content screening techniques were used to establish an allergic reaction-like in vitro detection method. RBL-2H3 cell degranulation model was used to determine the number of positive cells with cell-specific fluorescent dye FM4-64 labeled cell degranulation vesicles. The total number of cells was determined by labeling the nucleus with Hochest 3334, a nuclear-specific fluorescent dye, to calculate the drug-induced cell degranulation rate. In order to investigate the reliability of the high-content detection method, firstly, the positive drug Compound 48/80 was used to verify the consistency between the high content detection method and the traditional allergy mediator β-hexosaminidase release and mouse blue-skin detection method. High connotation detection method is highly consistent with the traditional detection methods, and the sensitivity is higher than the traditional detection methods; and then use 30 batches of clinical reports allergy symptoms Danhong injection to further verify the reliability of high content detection method, the results suggest that the high content of the detection results and clinical The report is highly consistent, and the high-content approach to some extent can overcome the injection of its own color interference. It can be seen that the high-content hypersensitivity test method is a sensitive, reliable, simple and high-throughput screening method. It is suitable for the evaluation of allergic reactions in pharmaceutical injection and can provide guidance for clinical safety medication.