中国中药杂志

2016, v.41(10) 1903-1909

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一种药物注射剂类过敏反应高内涵检测方法的初步建立
A high content screening method for detection of anaphylactoid reaction of injection formulations

王化龙;李振虎;吴红华;高秀梅;徐砚通;
WANG Hua-long;LI Zhen-hu;WU Hong-hua;GAO Xiu-mei;XU Yan-tong;Tianjin Key Laboratory of Modern Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine;Research and Development Center of Chinese Materia Medica,Tianjin International Joint Academy of Biotechnology and Medicine;

摘要(Abstract):

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。
Anaphylactoid reaction( AR) is the most common adverse reaction of injection formulations,however,there are obvious drawbacks in available methods for AR detection. A novel in vitro detection method for AR was established based on fluorescent labeling and high content screen( HCS) system in present study. With the use of RBL-2H3 cells degranulation model,positive cell count was determined with specific cellular membrane fluorescent dye FM4-64 labeling vesicle recycle,and total cells count was determined with specific nucleus fluorescent dye Hochest 3334,and then the ratio of cells degranulation after drug stimulation was calculated. In order to verify the reliability of this HCS method,positive drug Compound 48 /80 was first used to confirm the consistence of HCS method with the traditional β-hexosaminidase release test and the Evans blue staining ears test in mice. The results showed high consistence between HCS method and traditional testing methods,and the HCS method showed higher sensitivity than the other two tests. Then 30 samples of Danhong injection( DHI) with clinical allergy symptoms further were used to confirm the reliability of this HCS method. The HCS results showed high consistence with the clinical report,and the HCS method had the advantage in reducing the interference by drug color. Therefore,this HCS method is reliable,sensitive,simple and high-throughput method in detection of AR,applicable for the AR evaluation of injection formulations,and can provide guidance for safety of clinical application in clinical practice.

关键词(KeyWords): 类过敏反应;注射剂;高内涵;细胞脱颗粒
anaphylactoid reaction;injection;high content system;cell degranulation

Abstract:

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基金项目(Foundation): 国家“重大新药创制”科技重大专项(2013ZX09201020;2012ZX09101212);; 国家自然科学基金青年基金项目(81202798)

作者(Author): 王化龙;李振虎;吴红华;高秀梅;徐砚通;
WANG Hua-long;LI Zhen-hu;WU Hong-hua;GAO Xiu-mei;XU Yan-tong;Tianjin Key Laboratory of Modern Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine;Research and Development Center of Chinese Materia Medica,Tianjin International Joint Academy of Biotechnology and Medicine;

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