流式细胞术在小鼠局部淋巴结试验中的应用研究

摘要：目的　将流式细胞术应用到小鼠局部淋巴结实验（ｌｏｃａｌｌｙｍｐｈｎｏｄｅａｓｓａｙ，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 （犞／犞：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ＳＰＳＳ１１．５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（犘＜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
（ｆｌｏｗｃｙｔｏｍｅｔｒｙ，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。
关键词：局部淋巴结试验；小鼠；致敏性；刺激性；流式细胞术
中图分类号：Ｒ１１４；Ｒ９９４．６　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１４）０５ ０４７７ ０５

Application of flow cytometry on murine local lymph node assay

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.