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摘要：目的 建立体外高通量评价过敏反应模型，并对中药单体化合物进行筛选，从而确定其中的潜在过敏原。方法 建立稳定表达MrgX2的高通量药物筛选细胞模型。首先将重组质粒pmCherry-C1-MrgX2转染人胚肾HEK293细胞，通过G418筛选建立稳定表达人MrgX2的细胞株；再通过MrgX2特异性激动剂C48/80和拮抗剂2-APB检测细胞株的功能和稳定性，以Z因子确认高通量体系的稳定性和可靠性。采用上述模型对180个单体化合物进行筛选，以EC50、IC50、特异性及毒性确定该激动剂的功能性。结果 MrgX2细胞模型对特异性激动剂C48/80和特异性拮抗剂2-APB的EC50和IC50分别为2.7 ?g/mL和46.29 ?mol/L，MrgX2细胞模型第1代和第20代对激动剂的反应基本一致，该模型Z因子为0.78。筛选得到异莲心碱为阳性激动剂，其功能验证结果为：EC50为4.5 ?mol/L、IC50为39.47 ?mol/L，只能特异性激动MrgX2受体，且无细胞毒性。結论 采用钙流方法可成功构建基于过表达细胞系HEK293/MrgX2的体外高通量评价过敏反应模型，并且初步确定异莲心碱为潜在引发过敏反应的过敏原。
关键词：MrgX2；过敏反应评价方法；高通量；钙流检测；异莲心碱
DOI：10.3969/j.issn.1005-5304.2016.11.013
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2016）11-0051-06
Abstract： Objective To establish a high-throughput evaluation model for anaphylactic reactions； To screen and identify potential anaphylactogens from TCM monomeric compounds. Methods Cell model of stably expressed MrgX2 was established. Recombinate plasmid pmCherry-C1-MrgX2 was transfected to HEK293 to establish cell line for screening model. MrgX2 agonist and antagonist were used to identify the validation and stability of the cell line. A small library consisting of 180 compounds was profiled by using a cell-based calcium mobilization assay to find novel compounds targeting the MrgX2 receptor. EC50 test， IC50 test， specificity validation and cytotoxicity evaluation were carried out to detect the function of the positive agonist. Results The EC50 of C48/80 to MrgX2 model was 2.7 ?g/mL and the IC50 of 2-APB （evoked by 10 ?g/mL C48/80） was 46.29 ?mol/L. The first generation cell model of MrgX2 was similar to the 20th generation， and the Z factor of MrgX2 cell model was 0.78. In the primary screening for agonist， isoliensinine was identified as a novel agonist targeting receptor MrgX2 with an EC50 of 4.5 ?mol/L and IC50 of 39.47 ?mol/L. Moreover， isoliensinine was validated to activate MrgX2 receptor specifically without cytotoxicity. Conclusion A high-throughput evaluation method for anaphylactic reactions can be established in vitro through calcium mobilization assay. A potential anaphylactogen isoliensinine is identified and validated.
Key words： MrgX2； evaluation method for anaphylactic reactions； high throughput； calcium mobilization assay； isoliensinine
随着中药临床应用的日益增长，中药安全性“事件”时有发生[1]，尤其中药注射剂的临床安全性问题倍受关注[2]。目前过敏反应评价方法分为体内法和体外法。体内法存在主观性强、周期偏长、不适合大规模药物筛选等缺点[3]；体外法通常具有假阳性率高、人鼠种群的差异性等缺点[4]。因此，建立一种快速、准确的过敏反应检测方法势在必行。研究表明，MrgX2受体在过敏和慢性炎症中发挥着重要作用[5]。约翰霍普金斯大学研究人员确定MrgX2受体将是解决药物过敏反应的极其重要的靶点[6]。本研究建立基于过表达MrgX2的重组细胞系钙流高通量筛选方法，并应用所建模型对180个中药化合物进行筛选，以期对潜在致敏物质进行早期预测。
关键词：MrgX2；过敏反应评价方法；高通量；钙流检测；异莲心碱
DOI：10.3969/j.issn.1005-5304.2016.11.013
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2016）11-0051-06
Abstract： Objective To establish a high-throughput evaluation model for anaphylactic reactions； To screen and identify potential anaphylactogens from TCM monomeric compounds. Methods Cell model of stably expressed MrgX2 was established. Recombinate plasmid pmCherry-C1-MrgX2 was transfected to HEK293 to establish cell line for screening model. MrgX2 agonist and antagonist were used to identify the validation and stability of the cell line. A small library consisting of 180 compounds was profiled by using a cell-based calcium mobilization assay to find novel compounds targeting the MrgX2 receptor. EC50 test， IC50 test， specificity validation and cytotoxicity evaluation were carried out to detect the function of the positive agonist. Results The EC50 of C48/80 to MrgX2 model was 2.7 ?g/mL and the IC50 of 2-APB （evoked by 10 ?g/mL C48/80） was 46.29 ?mol/L. The first generation cell model of MrgX2 was similar to the 20th generation， and the Z factor of MrgX2 cell model was 0.78. In the primary screening for agonist， isoliensinine was identified as a novel agonist targeting receptor MrgX2 with an EC50 of 4.5 ?mol/L and IC50 of 39.47 ?mol/L. Moreover， isoliensinine was validated to activate MrgX2 receptor specifically without cytotoxicity. Conclusion A high-throughput evaluation method for anaphylactic reactions can be established in vitro through calcium mobilization assay. A potential anaphylactogen isoliensinine is identified and validated.
Key words： MrgX2； evaluation method for anaphylactic reactions； high throughput； calcium mobilization assay； isoliensinine
随着中药临床应用的日益增长，中药安全性“事件”时有发生[1]，尤其中药注射剂的临床安全性问题倍受关注[2]。目前过敏反应评价方法分为体内法和体外法。体内法存在主观性强、周期偏长、不适合大规模药物筛选等缺点[3]；体外法通常具有假阳性率高、人鼠种群的差异性等缺点[4]。因此，建立一种快速、准确的过敏反应检测方法势在必行。研究表明，MrgX2受体在过敏和慢性炎症中发挥着重要作用[5]。约翰霍普金斯大学研究人员确定MrgX2受体将是解决药物过敏反应的极其重要的靶点[6]。本研究建立基于过表达MrgX2的重组细胞系钙流高通量筛选方法，并应用所建模型对180个中药化合物进行筛选，以期对潜在致敏物质进行早期预测。