三维表皮模型Epikutis?参考品的研制

目的：考察体外重组三维（3D）表皮模型（Epikutis?）作为标准物质及用于替代模型皮肤刺激性检测方法的可行性。方法：利用由人源性角质形成细胞构建的体外重组3D表皮模型（Epikutis?）作为标准物质的原料，参照经济合作与发展组织439文件（OECD 439）：替代模型皮肤刺激性检测方法，选择组织活力和屏障功能指标，考察表皮模型的均匀性和稳定性，并通过多实验协作测定对其组织活力和屏障指数进行分析。其中，组织活力测定采用MTT试验，以阴性对照光密度值作为组织活力指标；屏障功能测定采用1% TritonX-100作用不同时间后，测定半数活性时间，作为屏障指数（ET₅₀）。结果：Epikutis?表皮模型（每批100多个）的均匀性良好，其样品间均匀性不确定度为：u_bb=0.013。短期稳定性（模拟保存运输条件下，运输箱内温度4~25℃放置24 h），组织活力（光密度，OD）为1.029，其短期稳定性不确定度为：u_s=0.018；ET₅₀的平均值为（2.08±0.12）h，其短期稳定性不确定度u_s=0.159，无明显活性及屏障功能降低趋势。协作测定结果：OD为1.033。其检测不确定度为：u_char=0.036；最终的合成不确定度（含u_bb、u_s和u_char）为0.043。ET₅₀的协作标定结果为1.70 h；最终的合成不确定度为0.159。选择10种OECD 439提供的参考物质种类，进行皮肤刺激性试验，其结果与OECD439发布的刺激性判定结果一致，符合预期结果。结论：Epikutis?表皮模型的组织活力：1.033±0.086，最终定值范围为：0.9≤OD≤1.2；ED₅₀：（1.70±0.32）h，最终定值范围为：1.4 h≤ET₅₀≤2.0 h。上述研究结果表明Epikutis?表皮皮肤模型满足OECD TG 439所推荐的替代模型皮肤刺激性检测方法的使用，可以用于化妆品、化学试剂、医疗器械等皮肤刺激性定性检测，也为纳米材料的透皮吸收及皮肤毒性研究提供了替代方法模型。

Objective: To investigate the feasibility of Epikutis?,a three-dimensional (3D) epidermal model as reference material and applicability of Epikutis? in in vitro skin irritation-reconstructed human epidermis test. Methods: The raw material used for reference material development was established based on the in vitro system of reconstructed human epidermis,which used human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model. This 3D epidermal model (Epikutis?) represents histology and cytoarchitecture of human skin. According to Organization for Economic Cooperation and Development (OECD) Test Guideline 439,tissue viability and barrier function of Epikutis? were selected as characteristic value of reference material,and using these items to evaluate the uniformity,stability and to definite characteristic value. The tissue viability was assayed by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT,detected optical density,OD) value of negative control,and the barrier function was measured by using 1% Triton X-100 for different time,and the half active time was detected as the barrier index (ET₅₀). Results: The Epikutis? models have good uniformity,and the uncertainty value was ubb=0.013 within over hundred model samples. The short-term stability was designed to mimic 24 h of preservation and transportation at 4-25℃,and the results showed that the tissue viability was 1.029 and uncertainty value was us=0.018,and the ET₅₀ value was (2.08±0.12) h and uncertainty value was us=0.159. Both the tissue viability and the ET₅₀ value have no significant decreases. Based on the results of collaborative examination of several laboratories,the average tissue viability was 1.033 and uncertainty value uchar=0.036,the final composite uncertainty (contains ubb、us and uchar) was 0.043;and the average ET₅₀ value was 1.70 h and the final composite uncertainty value was 0.159. The results of skin irritation tests on 10 chemicals selected from OECD 439 showed that the sensitivity was 100%,the results were consistent with the evaluation results published by the OECD439. Conclusion: The tissue viability of Epikutis? epidermal model was 1.033±0.086,and final range was 0.9 ≤ OD ≤ 1.2;and the ET₅₀ value was (1.70±0.32) h,and final range was 1.4 h ≤ ET₅₀ ≤ 2.0 h. The Epikutis? epidermal model is a suitable model that meets the application of OECD 439 in vitro skin irritation-reconstructed human epidermis test,and it has prospect to be used for qualitative detection skin irritation of cosmetic,chemicals,medical devices and also provide an alternative tool for investigating skin toxicity and permeability of nanomaterials.

[1] OECD Guideline for the Testing of Chemicals In Vitro Skin Irritation:Reconstructed Human Epidermis Test Method[S].2015:492
[2] SN/T 3948-2014化学品体外皮肤刺激性:重组人表皮试验[S].2014:1 SN/T3948-2014 Chemical-In Vitro Skin Irritation-Reconstructed Human Epidermis Test Method[S].2014:1
[3] SN/T 4577-2016化妆品皮肤刺激性检测重建人体表皮模型体外检测方法[S].2016:1 SN/T 3948-2014 Skin irritation test for cosmetics-Reconstructed human epidermal modelin vitro alternative method[S].2016:1
[4] 何立成,孙叶丹,瞿霖吉,等.重组人皮肤模型用于评价外用液体药用高密度聚乙烯瓶的原发性皮肤刺激性研究[J].中国现代应用药学,2018,35(6):801 HE LC,SUN YD,QU LJ,et al.Study on evaluating primary skin irritation of high-density polythylene bottles for topical liquid preparation using reconstructed human epidemis model[J].Chin J Mod Appl Phar,2018,35(6):801
[5] 何立成,张劲松,朱社敏,等.重组人皮肤模型用于评价药品包装材料原发性皮肤刺激质量标准的建立[J].中国药品标准,2018,19(6):442 HE LC,ZHANG JS,ZHU SM,et al.Establishment of the primary skin irritation test method of pharmaceutical packing materials with reconstructed human epidermis model[J].Drug Stand China,2018, 19(6):442
[6] OECD Guideline for the Testing of chemicals In vitro Skin Corrossion:Reconstructed Human Epidermis (RHE) Test Method[S].2015:431
[7] 张劲松,何立成,桑晶,等.EpiskinTM和Epikutis?模型在体外皮肤刺激性和腐蚀性检测应用中的比较[J].中国现代应用药学,2017,34(4):524 ZHANG JS,HE LC,SANG J,et al.Application comparison of EpiskinTM and Epikutis? Skin model in evaluating skin irritation and skin corrosion[J].Chin J Mod Appl Pharm,2017,34(4):524
[8] 唐洁,熊丽丹,李利.组织工程皮肤概况及其在化妆品检测中的应用进展[J].中国美容医学,2017,26(11):132 TANG J,XIONG LD,LI L,et al.Overview of tissue engineering skin and it's application in cosmetics test[J].Chin J Aesthet Med,2017,26(11):132
[9] ROGUET R,COHEN C,DOSSON KG,et al.Episkin,a reconstitutedhuman epidermis for assessing in vitro the irritancy of topicallyapplied compounds[J].Toxicol,1994,8(2):2831
[10] PORTES P,GRANDIDIER MH,COHEN C,et al.Refinement of the Episkin protocol for the assessment of acute skin irritation ofchemicals:follow-up to the ECVAM prevalidation study[J].Toxicolin Vitro,2002,16(6):765
[11] KANDOROVA H,LIEBSCH M,GENSCHOW E,et al.Optimisation of the Epiderm test protocol for the upcoming ecvam validationstudy on in vitro skin irritation tests[J].ALTEX,2004,21(3):107
[12] CHATTERJEE A,BABU RJ,KLAUSNER M,et al.In vitro and in vivocomparison of dermal irritancy of jet fuel exposure using Epi DermTM (EPI-200) cultured human skin and hairless rats[J].Toxicol Lett,2006,167(2):85
[13] HIKIMA T,KANEDA N,MATSUO,K,et al.Prediction of percutaneousabsorption in human using three-dimensional humanculturedepidermisLabCyte EPI-MODEL[J].Biol Pharm Bull,2012,35(3):362
[14] 刘洋,卢涛,周宙霖,等,HaCaT表皮模型作为皮肤刺激性体外替代实验的可行性研究[J].干细胞与组织工程,2017,31(10):1262 LIU Y.LU T.ZHOU ZL,et al.Study on feasibility of HaCaT epidermis model as an alternative to skin irritation in vitro[J].Chin J Repar Reconstre Surg,2017,31(10):1262
[15] 姜珊,陈亮,吴美玉,等.金核/银壳纳米棒在三维皮肤模型中的透皮行为和对组织活力的影响[J].药物分析杂志,2019,39(3):377 JIANG S,CHEN L,WU MY,et al.Transdermal behavior of gold/silver shell nanorods in three-dimentional epidermal model and it's effect on tissue activity[J].Chin J Pharm Anal,2019,39(3):377
[16] GERTRUDE-MILIA E,HANS R,RODGER C,et al.In vitro safety testing strategy for skin irritation using the 3D reconstructed human epidermis[J].J Biochem,2009,46(2):165