流式细胞术在小鼠局部淋巴结试验中的应用研究

Application of flow cytometry on murine local lymph node assay

目的将流式细胞术应用到小鼠局部淋巴结实验（local lymph node assay,LLNA）中,期望建立既可检测化学物致敏性也可同时检测刺激性的方法。方法受试物分为溶剂对照[AOO（丙酮∶橄榄油=4∶1）]、阴性对照（NaCl）、致敏阳性物[己基肉桂醛（HCA）、2,4-二硝基氯苯（DNCB）、2-氨基酚（2-APC）]、刺激阳性物[KOH、十二烷基硫酸钠（SLS）]。采用8-10周龄16-22g的雌性Balb/c小鼠76只,随机分为19个受试物剂量组,每剂量组4只小鼠。每种受试物分别设低、中、高3个剂量组,检测浓度分别为NaCl（100g/L、200g/L、300g/L）、HCA（V/V：3.3%、10.0%、30.0%）、DNCB（2.5g/L、5.0g/L、10.0g/L）、2-APC（12.5g/L、25.0g/L、50.0g/L）、KOH（25g/L、50g/L、100g/L）、SLS（50g/L、100g/L、200g/L）。于试验第1、2、3天将配制后不同浓度受试物稀释液以每耳25μl/d均匀涂布于小鼠双侧耳后。检测小鼠耳缘厚度、耳引流淋巴结重量、B220＋（CD45R）亚群淋巴细胞百分数等指标。采用SPSS 11.5分析软件,对数据型指标进行方差分析,并对有差异的个体再行LSD两两比较,并根据刺激指数对受试物致敏性大小进行分级。结果与溶剂对照比较中高剂量的SLS和KOH均可引起小鼠耳缘厚度增加（P〈0.01）。可判定SLS、KOH对皮肤有刺激性。DNCB、HCA各剂量组和2-APC高剂量组刺激指数均大于3,可以判断该3种化学物为皮肤致敏物。根据SI计算EC3的值,对致敏物进行分类：DNCB（0.196）为强致敏物,HCA（1.556）和2-APC（3.156）为中等致敏物。结论应用流式细胞术（flow cytometry,FCM）检测B220＋（CD45R）亚群淋巴细胞百分数作为致敏性观察指标的改良小鼠淋巴结实验,结合耳缘厚度检测可实现同时评价化学物的皮肤致敏性和刺激性。

Objective To explore the potential application of flow cytometry in murine local lymph nodes assay（LLNA）for testing both chemical sensitization and irritation. Methods Test substances used in the study included solvent control（AOO）,negative control（NaCl）,sensitizers（HCA,DNCB and 2-APC）and irritants（KOH,SLS）,and each testing material was prepared at low,medium and high doses（NaCl 100,200,300g/L,HCA 3.3%,10%,30%,DNCB 2.5,5.0,10.0g/L,2-APC 12.5,25.0,50.0g/L,KOH 25,50,100g/ml,SLS 50,100,200g/L）.76 female Balb/c mice aged 8-10 weeks were evenly divided into 19 testing groups and was coated with 25μl per ear of different dosage of testing material around the back of both ears in the first 3days.The thickness of ear,ear drainage lymph nodes weight and lymphocyte percentage of B220＋（CD45R）subgroup were measured and determined.Data were processed by ANOVA,and were further compared and analyzed by LSD if the difference was significant.The sensitization was graded according to the stimulating index. Results Significant increases in ear marginal thickness were found in mice at medium and high dosage groups of KOH and SLS compared to mice in solvent control（P〈0.01）,which was considered as the result of irritation.Meanwhile,the stimulating index of high dose 2-APC and 3testing dosages of HCA and DNCB were all higher than 3,indicating that they were all chemical sensitizers.According to the value of EC3,DNCB（0.196）was classified as strong sensitizer and HCA（1.556）and 2-APC（3.156）as medium sensitizers. Conclusions When combined with mice ear marginal thickness,lymphocyte percentage of B220＋（CD45R）determined by flow cytometry as observed marker for sensitization in reformed LLNA can be used to evaluate both sensitization and irritation at the same time.