中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (2): 127-128.

• 技术与方法 • 上一篇    下一篇

体外3T3成纤维细胞中性红摄取试验对光毒物质的评价

吕中明1,俞萍2,王民生2,陈斌2   

  1. 1. 江苏省疾病预防控制中心
    2. 南京医科大学第一附属医院皮肤科
  • 收稿日期:2008-11-17 修回日期:2009-09-18 出版日期:2010-02-15 发布日期:2012-03-30
  • 通讯作者: 吕中明 E-mail:lvzhming@hotmail.com
  • 基金资助:
    江苏省预防医学科研基金(Y200423);省级基金（编号）

Photoxicity evaluation using in vitro 3T3 NRU phototoxicity test

  • Received:2008-11-17 Revised:2009-09-18 Online:2010-02-15 Published:2012-03-30

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摘要： 目的 建立Balb/c 3T3成纤维细胞中性红摄取试验(3T3-NRU PT)，研究其作为化学品安全性评价中替代动物光毒性试验的可行性。方法 ⑴Balb/c 3T3细胞经过UVA不同照射剂量照射后，用中性红摄入法测定不同照射剂量的细胞活力；⑵选取十二烷基硫酸钠(SDS)，8-甲氧基补骨脂(8-MOP)，盐酸氯丙嗪(CPZ)作为受试物,按照OECD“化学品测试导则432”的推荐方法，将Balb/c 3T3 成纤维细胞接种于96 孔细胞培养板，每个样品接种2块培养板，染毒1h后,随机选择一块培养板用紫外照射仪（选UVA光源365nm)进行照射(+Irr),照射剂量为5J/cm2,另一平行板置于黑暗中不照射(-Irr)作为对照。孵育24h后换中性红培养液培养3h,加入中性红解吸附液，于540nm处酶标仪测定NRU的吸光度值。根据测定的吸光度值, 使用PHOTO32 软件分别建立平均光效应(MPE值) 和光刺激因子(PIF值) 预测模型,以判断所测物质是否具有光毒性。 结果 ⑴ Balb/c 3T3细胞经紫外线照射后, 随着照射剂量的增加,细胞存活率逐渐降低。照射剂量为5J/cm2时细胞的存活率高于90%,但剂量达7.5J/cm2时细胞存活率就低于80%，显示出一定的细胞毒性，故照射剂量应不高于5J/cm2为宜；⑵ 3T3-NRU PT试验测得SDS、8-MOP、CPZ的PIF值分别为0.89、22.81和17.60；MPE值分别为0.001、0.706和0.502,按该试验阈值分类方法, SDS为无光毒性物质，8-MOP、CPZ均具有光毒性。结论 本实验室成功地建立了3T3-NRU PT，并在照射剂量控制等方面进行了探索，从而为化学品安全性评价提供依据。

关键词: 皮炎，光毒性, 成纤维细胞

Abstract: Objective To establish and improve Balb/c 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU-PT) method for detecting potential phototoxicity in the safety assessment. Method According to the guidelines for the testing of chemicals 432 (OECD), Balb/c 3T3 cell was incubated in 96-well plate, SDS, 8-MOP and CPZ were detected as the test substances. Biospectra system (France) was used as the UV radiation source, and the wavelength was 365nm. The potential phototoxicity was evaluated in a prediction model in which either the photo irritation factor (PIF) or the mean photo effect (MPE) were determined by Photo32 (Version 2.0). Result (1) when the irradiated dosage attained 5 to 20J/cm2, the relative survival rate gradually reduced; (2) The results of 3T3-NRU PT showed the value of PIF and MPE were 0.89、22.81、17.60 and 0.001、0.706、0.502 for SDS, 8-MOP and CPZ, respectively, according to the test threshold, SDS has no phototoxicity, 8-MOP and CPZ has obvious phototoxicity. Conclusion The 3T3-NRU PT was successfully established in our laboratory, and we took some improvements in the UV source choice and irradiation time, it may be a good method for evaluating the potential phototoxicity in safety assessment in future.

Key words: fibroblasts