小鼠局部淋巴结实验在红花注射液致敏性研究中的应用

Application of the Mouse Popliteal Lymph Node Assay in the Study of Sensitization Potential of Safflower Injection

目的建立小鼠局部淋巴结实验模型检测红花注射液的致敏性,探讨其用于评价中药注射剂致敏的可行性。方法BALB/c小鼠90只,随机分为9组,即3个阳性对照组(分别给予2,4,6-三硝基苯磺酸、链脲佐菌素、D-青霉胺),1个阴性对照组(给予磷酸盐缓冲液)和5个供试品组(分别给予5批红花注射液),每组10只,在小鼠左后肢足趾部注射相应试药1次,每只50μL,第7天处死动物,采用小鼠局部淋巴结实验方法(LLNA)测定腘窝淋巴结质量指数(WI)及细胞指数(CI),采用流式细胞术法测定淋巴细胞亚群,观察各供试品组与对照组的差别。采用豚鼠全身主动过敏实验(ASA)对5批供试品进行同步检测,并与小鼠局部淋巴结实验进行比较。结果小鼠局部淋巴结实验结果显示5批红花注射剂中,仅1批WI有增加的趋势,且T淋巴细胞比例升高,提示这批供试品可能引起淋巴结的增大和T淋巴细胞的增殖,具有一定的致敏潜在风险,而其他批次供试品指标的变化均差异无统计学意义;ASA结果显示5批红花注射剂均未见变态反应。两种方法的结果基本一致。结论在该实验条件下,初步判定小鼠局部淋巴结分析实验可以作为ASA的一种替代方法评价红花注射液致敏的潜能。

Objective To establish a mouse model for local lymph node test to detect the sensitization of safflower injection,and to explore its feasibility for evaluating the sensitization of traditional Chinese medicine injection.Methods Ninety BALB/c mice were randomly divided into 9 groups(10 in each group):three positive control groups(2,4,6-trinitrobenzenesulfonic acid,streptozotocin,and D-penicillamine),one negative control group(phosphate buffered solution)and five test groups(5 batches of safflower injection).The mice were immunized with the test materials of at the left hind foot,50μL per mouse.The animals were sacrificed on the 7th day after injection.The weight index(WI)and cell index(CI)of bilateral popliteal lymph node were measured by local lymph node test(LLNA),and the lymphocyte subsets were detected by flow cytometry.The difference between each test group and the control group was observed.The guinea pig systemic active allergy test(ASA)was used to test the 5 batches of the test samples in parallel,and the results were compared with the mouse local lymph node assay.Results The results of local lymph node assay showed that only one batch of safflower injection had an increasing trend in WI and the proportion of T lymphocytes increased,which suggested that the test samples might cause the enlargement of lymph nodes and the proliferation of T lymphocyte.There was a potential risk of sensitization.The ASA results showed no allergic reactions in 5 batches of injections.The results of the two methods are basically the same.Conclusion Under the experimental conditions,the preliminary determination of mouse LLNA can be used as an alternative method of ASA to evaluate the sensitization potential of safflower injection.