RBL-2H3细胞体外试验评价药物类过敏的方法学研究和适用性考察

Methodological Study of Evaluation of Drug Allergy Using RBL-2H3 Cells in vitro

目的：RBL-2H3细胞用于检测药物类过敏的方法学研究,对评价类过敏反应的适用性进行考察.方法：采用MTT试验研究RBL-2H3细胞的密度、生长时间等基本生长特性,以化合物48/80（C48/80）为工具药物对其最佳给药浓度、作用时间及检测指标进行优化,以聚山梨酯（吐温80）、中华眼镜蛇毒作适用性考察,对血塞通、脉络宁注射液类过敏反应进行初步评价.结果：RBL-2H3细胞试验作为药物类过敏反应体外检测方法,最佳培养时间为24 h,最佳种植密度为（0.5 ～2.0）×10^5/mL.实验选用台氏液为反应溶媒,其C48/80致RBL-2H3细胞脱颗粒试验以25 ～50 mg·L^-1质量浓度,反应30 min为优选条件.C48/80可使细胞上清β-己糖苷酶、组胺浓度升高,呈现良好的“量-时-效”关系;β-己糖苷酶、组胺释放率与药物剂量呈高度正相关（r =0.984,0.940;P =0.000,0.000）,两指标之间相关性非常显著（r=0.957;P=0.000）.适用性考察显示,吐温80、中华眼镜蛇毒素在临床或高于临床浓度条件下均具有致RBL-2H3细胞脱颗粒作用;中药注射剂血塞通注射液存在潜在发生类过敏风险,脉络宁注射液类过敏反应阴性.结论：RBL-2H3细胞作为药物类过敏的体外试验方法具有灵敏、快速、方便的特点,适用于包括中药注射剂在内的药物类过敏反应的检测和评价,对于存在明显色质的受试物,建议以ELISA法指标组胺检测结果为准,最终的确证还需结合整体动物试验.

Objective： The methodology study was conducted in RBL-2H3 cells allergy test, and its applicability into evaluating anaphylactoid reactions was studied. Method： The grown characteristics of RBL-2H3 cells were observved, then compound 48/80 （C48/80） was used to optimize the experimental conditions such as cell density, concentration of dosage, testing indexes. The application of the method was studied though testing two reported allergic reaction drugs, which was used to evaluate two herbs injections. Result： As a vitro test to detect drug allergic reaction, the optimum planting density of RBL-2H3 test was （0.5-2.0） ×10^5/mL, optimal incubation time was 24 h, optimal drug interaction medium was Tyrode＇ s solution; the optimal condition of C48/80 induced RBL-2H3 cell degranulation test was 25-50 mg ·L^-1 concentration and response 30 min; C48/80, Tween- 80 and Chinese cobra toxin could induce RBL-2H3 cell degranulation and the increase of cell supernatant β-glucosidase and histamine concentrations, which showed a good ＇ quantity-time-effect＇ relationship. For Xuesaitong the potential anaphylactoid risk was found. Conclusion： As a in vitro test lbr drug anaphylactoid, RBL-2H3 cell method is sensitive, rapid and convenient, it suits the anaphylactoid testing of drugs including TCM injections. For non-ferrous subjects content, it is suggested that the index of histamine ELISA method, confirm at last still need to be combined with experiment in vivo.