以IL-8为评价指标的THP-1细胞光致敏评价方法的确定和验证  

Identification and validation of THP-1 cell photoallergy evaluation method with IL-8 as evaluation index

作　　者：董建欣 黄舒佳 王宇 孙旭[2] 赵华琛 祝清芬 刘丽 李波 DONG Jianxin;HUANG Shujia;WANG Yu;SUN Xu;ZHAO Huachen;ZHU Qingfen;LIU Li;LI Bo(Key Laboratory of Generic Drug Research and Evaluation,Shandong Institute for Food and Drug Control,Jinan 250101,China;Key Laboratory of Beijing for Nonclinical Safety Evaluation Research of Drugs,National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176,China;Center for Drug Evaluation,National Medical Products Administration,Beijing 100022,China)

机构地区：[1]山东省食品药品检验研究院国家药品监督管理局仿制药研究与评价重点实验室,山东济南250101 [2]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176 [3]国家药品监督管理局药品审评中心,北京100022

出　　处：《药物评价研究》2022年第1期10-19,共10页Drug Evaluation Research

基　　金：国家“重大新药创制”科技重大专项资助项目“创新药物非临床安全性评价研究关键技术”(2018ZX09201017)。

摘　　要：目的确定并验证以白细胞介素-8(IL-8)作为评价指标的THP-1细胞光致敏体外评价方法。方法将THP-1细胞与19种光致敏剂、4种光刺激剂、2种皮肤致敏剂、1种皮肤刺激剂和阴性受试物分别孵育24 h,在光照射(1.7 mW·cm^(−2),50 min)或避光处理后,细胞换液放入培养箱内继续培养5 h。光刺激剂需在正式光照射前进行预照射(光照30 min,然后避光15 min)处理。用Luminex液相芯片检测技术测定细胞培养上清和细胞裂解液中IL-8和肿瘤坏死因子-α(TNF-α)的含量并统计分析,进一步确定评价光致敏的细胞因子指标,并进行方法验证。结果与非照射组比较,13种光致敏剂均引起照射组THP-1细胞IL-8总含量显著增加(P<0.01),阿伏苯宗为非照射组的1148~2269倍,其余12种为非照射组的6.7~195.1倍;光刺激剂经光照射后也可引起细胞分泌IL-8显著增加(P<0.01),但经过预照射处理后,IL-8的含量相比直接照射组显著下降(P<0.01)。未经光照射条件下,皮肤致敏剂即可引起THP-1细胞分泌IL-8比对照组显著增加(P<0.01),光照后IL-8含量虽也较非照射组显著增加(P<0.01),但增加的幅度小于皮肤致敏剂本身引起IL-8变化的幅度。在光照条件下,皮肤刺激剂、阴性受试物均可引起细胞分泌IL-8较非照射组显著性增加(P<0.05),但增加的幅度较小,与对照组相似。以上受试物引起光照前后细胞TNF-α变化的幅度均与对照组相近。以IL-8为评价指标的THP-1细胞光致敏评价方法检测光致敏剂的准确性、特异性和灵敏度分别是77.8%、100.0%、68.4%,并且该方法具有良好的重复性。结论确定了THP-1细胞光致敏评价方法的细胞因子标志物评价指标为IL-8,判定标准为:①UVA照射后THP-1细胞中IL-8含量比非照射组显著增加;②UVA照射组与非照射组IL-8含量比值≥6.5;③当上述两条均满足时,对受试物进行UVA预照射处理,预处理照射组IL-8的含量与直接照射组(未经预照�Objective An in vitro evaluation method for photosensitization of THP-1 cells using interleukin(IL)-8 was established and validated.Methods THP-1 cells were treated with 19 photosensitizers,four photoirritants,two skin sensitizers,one skin irritant and one negative subject respectively.After solar radiation(1.7 mW·cm^(−2),50 min)or non-radiation treatment,cells were placed into the incubator for further culture for 5 h.Photostimulant should be pre-irradiated(30 min of light,then 15 min of avoid light)before formal light exposure.The contents of cytokines IL-8 and tumor necrosis factor-α(TNF-α)in cell culture supernatant and cell lysis fluid were determined by Luminex liquid chip detection technology and statistically analyzed,to further determine the cytokine indexes for evaluating photosensitization and verify the method.Results Twelve photosensitizers caused significant increases in IL-8 content in THP-1 cells in the irradiation group(P<0.01),which was 6.7 to 195.1 times of that in the non-irradiation group.Avobenzone was 1148 to 2269 times higher than that of the non-irradiated group.The four photoirritants also induced significant increases in IL-8 secretion after light irradiation(P<0.01),but after pre-irradiation,the IL-8 contents were significantly decreased compared with that in the direct irradiation group(P<0.01).The two skin sensitizers induced THP-1 cells to secrete significantly more IL-8 than the control group without light irradiation(P<0.01).Although the content of IL-8 after light irradiation was also significantly increased compared with that before irradiation(P<0.01),the amplitude of increase was smaller than that caused by skin sensitizer itself without light exposure.The skin irritant and negative subject could significantly increase the secretion of IL-8 compared with non irradiation group,but the increase amplitude were small,and were lower than the minimum change amplitude of IL-8 content induced by photoallergy agents.The change amplitudes of TNF-αlevel between irradiation and non

关 键 词：光致敏 THP-1细胞 白细胞介素 肿瘤坏死因子-Α 方法验证 

分 类 号：R965.1[医药卫生—药理学]