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目的改进小鼠局部淋巴结实验(LLNA),建立既可检测化学物致敏性也可同时检测刺激性的LLNA替代方法。方法选取一种阴性物质:4-氨苯甲酸;三种致敏阳性物:2,4-二硝基氯苯(DNCB)、己基肉桂醛(HCA)和2-氨基酚(2-APC);以及两种刺激阳性物:氢氧化钾(KOH)、十二烷基硫酸钠(SLS)。按常规LLNA方法对雌性Balb/c小鼠进行染毒,测量小鼠耳缘厚度并称量耳廓重量,于第6天获取耳引流淋巴结进行称重,制备单细胞悬液计数并用CCK-8试剂盒检测淋巴细胞增殖活性。结果受试剂量氢氧化钾、十二烷基硫酸钠和2,4-二硝基氯苯在0.5%以上剂量,可引起耳缘厚度和耳廓重量增加,与对照组相比差异有显著性(P<0.05),可判定为对皮肤有刺激性,2-氨基酚和己基肉桂醛均无刺激性。三种致敏物在致敏试验中均呈阳性,但三指标的敏感性略有不同。己基肉桂醛,2,4-二硝基氯苯,2-氨基酚均可引起淋巴结重量增加和淋巴细胞计数增加(P<0.05);中剂量以上组己基肉桂醛和2,4-二硝基氯苯和高剂量2-氨基酚的CCK-8检测均见淋巴细胞增殖明显。结论以耳缘厚度和耳廓重量作为刺激性观察指标,以淋巴结重量和淋巴细胞增殖作为致敏性观察指标的改良小鼠淋巴结实验可有效地同时评价化学物的皮肤致敏性和刺激性。
Objective To improve the LLNA in mice and establish an alternative method of LLNA that can both detect allergenicity of chemicals and detect irritation simultaneously. Methods A negative sample of 4-aminobenzoic acid and three sensitized positive samples of 2,4-dinitrochlorobenzene (DNCB), hexylcinnamaldehyde (HCA) and 2-aminophenol As well as two stimulus-positives: potassium hydroxide (KOH), sodium lauryl sulfate (SLS). The female Balb / c mice were exposed to the conventional LLNA method. The thickness of the ear marginalis was measured and the pinna was weighed. Ear draining lymph nodes were harvested on day 6 and weighed. Single cell suspensions were counted and stained with CCK-8 Kits were tested for lymphocyte proliferation activity. Results The dosage of potassium hydroxide, sodium dodecyl sulfate and 2,4-dinitrochlorobenzene at 0.5% dose increased the thickness of the ear and the auricle weight, which was significantly different from the control group (P <0.05), can be judged as skin irritant, 2-aminophenol and hexylcinnamaldehyde are not irritating. Three allergens in the sensitization test were positive, but the sensitivity of the three indicators are slightly different. Hexylcinnamaldehyde, 2,4-dinitrochlorobenzene and 2-aminophenol all increased lymph node weight and lymphocyte count (P <0.05). The middle dose of hexyl cinnamic aldehyde and 2,4-dinitro Chlorobenzene and high-dose 2-aminophenol CCK-8 test showed lymphocyte proliferation significantly. Conclusion The thickness of ear rim and auricle weight as the irritant observation index, the lymph node weight and lymphocyte proliferation as the allergen observation index improved mouse lymph node test can effectively evaluate the chemical skin sensitization and irritation.