基于BALB/c小鼠的局部淋巴结试验用于皮肤致敏性评价的实验研究

目的：用BALB/c小鼠建立基于掺入溴脱氧尿嘧啶核苷（BrdU）和酶联免疫吸附测定（ELISA）的小鼠局部淋巴结试验（LLNA）改良方法（LLNA：BrdU-ELISA），并对该方法进行评价。方法：试验设溶剂对照组（丙酮/橄榄油、丙酮、二甲基亚砜或蒸馏水），阳性对照组（25%己基肉桂醛）和受试物组（选择22种化学物和5种化妆品，配成1~3个不同浓度），每组4只小鼠。每天上午9：00在小鼠双耳背部分别涂抹受试物25 μL，连续染毒3 d，第4天不作处理，第5天腹腔注射BrdU标记液0.5 mL。第6天称量耳廓质量，分离颌下淋巴结称取质量并制成单细胞悬液，用ELISA试剂盒测定淋巴细胞增殖。结果：50%己基肉桂醛、5%二氯化钴、10%乳酸、25%乳酸、50%乳酸和2号染发剂引起耳廓质量显著增加（P < 0.05或P < 0.01），为刺激阳性，其他化学物和化妆品为刺激阴性；15种化学物（2，4-二硝基氯苯、丁子香酚、异丁子香酚、3-氨基苯酚、反式肉桂醛、己基肉桂醛、香叶醇、对苯二酚、水杨酸己酯、甲醛、戊二醛、六水硫酸镍、二氯化钴、重铬酸钾、乳酸）和2号染发剂为致敏阳性（SI>1.6），其他化学物或化妆品为致敏阴性。该方法阳性预测率为93.3%，阴性预测率为100%，假阳性率为14.3%，假阴性率为0，灵敏度为100%，特异性为85.7%、准确度为95.2%。结论：LLNA：BrdU-ELISA方法用BALB/c小鼠可较好地评价化学物/化妆品的刺激性和致敏性，可在化妆品安全性评价中发挥重要作用。

Experimental study based on the local lymph node assay in BALB/c mice for skin sensitization evaluation

OBJECTIVE: To establish and evaluate a modified local lymph node assay incorporated with bromodeoxyuridine and enzyme-linked immunosorbent assay. METHODS: Female BALB/c mice were organized into several groups of 4 mice per group:solvent control (AOO, ACE, DMSO or distilled water), positive control (25% hexyl cinnamaldehyde) and test substance (22 chemicals and 5 cosmetics were selected to form 1-3 different concentrations) groups. The dorsal sides of both ears of mice were treated with 25 μL/ear of test solutions at 9:00 am on 3 consecutive days with no treatment on day 4. BrdU solution was injected interperitoneally on day 5 with 0.5 mL per mouse. On day 6, ear punches were collected and weighted. The bilateral draining auricular lymph nodes were excised and made into single cell suspensions from which lymphocyte proliferations were measured using the BrdU ELISA kit. RESULTS: Ear punch weights from mice treated with 50% hexyl cinnamic aldehyde,5% cobalt dichloride,10%,25%,50% lactic acid and No.2 hair dye were increased significantly (P < 0.05 or P < 0.01),which might be considered as irritants. The others were negative. For LLNA, 15 chemicals (2,4-dinitrochloro-benzene, eugenol, isoeugenol, 3-aminophenol, transcinnamic aldehyde, hexyl cinnamic aldehyde, geraniol, hydroquinone, hexyl salicylate, formaldehyde, glutaraldehyde, nickel sulfate, cobalt dichloride, potassium dichromate, lactic acid) and No.2 hair dye were positive (SI>1.6),and the others were negative. The method was evaluated with positive prediction rate 93.3%, negative prediction rate 100%,false positive rate 14.3%,false negative rate 0,sensitivity 100%,specificity 85.7% and accuracy 95.2%. CONCLUSION: The LLNA:BrdU-ELISA assay using BALB/c mice appears to be useful for predicting sensitization and irritancy potential,and for safety evaluation of cometics.