以细胞表面标志物为研究指标的光致敏体外评价方法
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摘要
目的:建立一种以细胞表面标志物作为评价指标的光致敏体外评价方法。方法:应用THP-1细胞,分别与光致敏剂、光刺激剂、皮肤致敏剂、皮肤刺激剂、阴性受试物等10余种受试物进行孵育,紫外照射和非照射条件下分别检测细胞表面标志物CD54和CD86的表达变化,确定合适的辐照强度、受试物剂量和光照后检测时间点,并确定评价光致敏的敏感指标和初步判定标准。结果:光致敏剂与THP-1细胞孵育并经紫外照射后,表达CD54的THP-1细胞百分比及平均荧光强度MFI比非照射组显著增加,相对荧光强度RFI在1. 4以上。光致敏剂未引起CD86表达的明显变化。其他受试物模型在照射前后CD54的MFI未见显著变化或变化程度明显低于光致敏剂。皮肤致敏剂引起的细胞表面标志物变化特点与光致敏剂明显不同。结论:THP-1细胞表面标志物CD54对于评价化合物光致敏性具有良好的灵敏度和特异性,本研究建立了一种应用细胞表面标志物进行光致敏性评价的体外方法,初步确定了评价指标和判定标准。
Objective: To establish an in vitro photosensitization evaluation method using cell surface markers as evaluation index. Methods: THP-1 cells were incubated with more than 10 test substances separately,including photoallergy agents,photo irritant,skin sensitizer,skin irritant,and negative control. Then the expression changes of cell surface markers of CD54 and CD86 were detected under ultraviolet radiation and non-radiation conditions respectively. The appropriate irradiation intensity,dose levels of the test substances and the detection time points after illumination were determined. The sensitive end points for photosensitization evaluation were screened and the preliminary judgement criteria was set up. Results: After incubation of photoallergy agents with THP-1 cells and exposure to UVA irradiation,the percentage of THP-1 cells expressing CD54 and the mean fluorescence intensity MFI increased significantly compared with the non-irradiation group,and the relative fluorescence intensity RFI was above 1. 4. The photoallergy agents did not cause obvious changes in CD86 expression. No significant changes in CD54 MFI were found post UVA irradiation for the other test articles,or the degree of change was significantly lower than that of the photoallergy agent. The change characteristics of the cell surface markers induced by skin sensitizers were distinctively different from those of photosensitization agents. Conclusion: THP-1 cell surface marker CD54 has good sensitivity and specificity for evaluating the photosensitization of compounds. In this study,an in vitro method for photoallergy evaluation using cell surface markers was established,and the evaluation index and judgement criteria were preliminarily determined.
Objective: To establish an in vitro photosensitization evaluation method using cell surface markers as evaluation index. Methods: THP-1 cells were incubated with more than 10 test substances separately,including photoallergy agents,photo irritant,skin sensitizer,skin irritant,and negative control. Then the expression changes of cell surface markers of CD54 and CD86 were detected under ultraviolet radiation and non-radiation conditions respectively. The appropriate irradiation intensity,dose levels of the test substances and the detection time points after illumination were determined. The sensitive end points for photosensitization evaluation were screened and the preliminary judgement criteria was set up. Results: After incubation of photoallergy agents with THP-1 cells and exposure to UVA irradiation,the percentage of THP-1 cells expressing CD54 and the mean fluorescence intensity MFI increased significantly compared with the non-irradiation group,and the relative fluorescence intensity RFI was above 1. 4. The photoallergy agents did not cause obvious changes in CD86 expression. No significant changes in CD54 MFI were found post UVA irradiation for the other test articles,or the degree of change was significantly lower than that of the photoallergy agent. The change characteristics of the cell surface markers induced by skin sensitizers were distinctively different from those of photosensitization agents. Conclusion: THP-1 cell surface marker CD54 has good sensitivity and specificity for evaluating the photosensitization of compounds. In this study,an in vitro method for photoallergy evaluation using cell surface markers was established,and the evaluation index and judgement criteria were preliminarily determined.
引文
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[2]MORITA O,OGURA R,HAYASHI K,et al.Safety studies of pseudo-ceramide SLE66:acute and short-term toxicity[J].Food Chem Toxicol,2009,47(4):669-673.
[3]孔雪,赵华,唐颖.皮肤模型在化妆品功效评价中的应用研究进展[J].日用品化学工业,2017,47(4):228-236.
[4]李航,王晖.朗格汉斯细胞在皮肤免疫学中的研究进展[J].中国皮肤性病学杂志,2014,28(3):304-306.
[5]CHIPINDA I,RUWONA TB,TEMPLETON SP,et al.Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and pro-hapten sensitizers[J].Toxicol,2011,280(3):135-143.
[6]LIU X,XUE Y,DING T,et al.Enhancement of proinflammatory and procoagulant responses to silica particles by monocyte-endothelial cell interactions[J].Part Fibre Toxicol,2012,9(1):1-15.
[7]SHINOBU K,TOSHIHIKO S,YOSHIO K,et al.Mechanism for6-Methylcoumarin Photo allergenicity[J].Toxicol Appl Pharm,1985,81(2):295-301.
[8]KARSCHUK N,TEPE Y,GERLACH S,et al.A novel in vitro method for the detection and characterization of photosensitizers[J].Plos One,2010,5(12):58.
[9]MASATOSHI H,MORIHIKO H,MIE S,et al.Development of an in vitro photosensitization assay using human monocyte-derived cells[J].Toxicol In Vitro,2009,23(5):911-918.
[10]LEONARD F,KALIS B.Photoprotection by drugs[J].Rev Prat,1992,42(11):1375-1376.
[11]ONOUE S,OHTAKE H,SUZUKI G,et al.Comparative study on prediction performance of photosafety testing tools on photoallergens[J].Toxicol In Vitro,2016,33(1):147-152.
[12]International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use.ICH guideline S10 Guidance on photosafety evaluation of pharmaceuticals[S].2014.
[13]RYOSUKE HINO,HIROSHI ORIMO,KENJI K,et al.Evaluation of photoallergic potential of chemicals using THP-1 cells[J].J Dermatol Sci,2008,52(2):140-143.
[14]OECD.Guideline For The Testing Of Chemicals,NO 442E:In Vitro Skin Sensitisation:human Cell Line Activation Test(hCLAT)[S].2016.
[15]BOCK S,SAID A,MULLER G,et al.Characterization of reconstructed human skin containing Langerhans cells to monitor molecular events in skin sensitization[J].Toxicol In Vitro,2018,46(1):77-85.
[16]RYAN CA,HULETTE BC,GERBERICK GF.Approaches for the development of cell-based in vitro methods for contact sensitization[J].Toxicol In Vitro,2001,15(1):43-55.
[17]许可,赵瀛兰,王莉,等.用局部淋巴结试验(LLNA)预测中药皮肤致敏性的研究[C].中国毒理学会中药与天然药物毒理专业委员会第一次(2016年)学术交流大会,2016.
[18]NUKADA Y,ASHIKAGA T,SAKAGUCHI H,et al.Predictive performance for human skin sensitizing pootential of the human cell line activation test(h-CLAT)[J].Contact Dermatitis,2011,65(6):343-353.
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