以CD54为评价指标的THP-1细胞光致敏体外评价方法的确定和验证

Identification and validation for in vitro photoallergy evaluation method of THP-1 cell with CD54 as evaluation index

目的确定并验证以细胞表面标志物CD54作为评价指标的光致敏体外评价方法。方法将THP-1细胞与多种光致敏剂、光刺激剂、皮肤致敏剂、皮肤刺激剂和阴性受试物分别孵育,在光照射或避光处理后,用流式细胞仪测定细胞表面标志物CD54和CD86的表达水平并统计分析,进一步确定具体的评价指标,并对该方法的准确性、特异性、灵敏度、重复性进行验证。结果19种光致敏剂中有15种引起照射组THP-1细胞表达CD54的平均荧光强度(MFI)较非照射组显著增加(P<0.05、0.01),且照射组细胞表达CD54的相对荧光强度(RFI)值均在1.5以上。光刺激剂经光照射后也可引起细胞表达CD54的MFI显著增加(P<0.01),但经过预照射处理后,CD54的表达水平较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细胞表达CD54和CD86的MFI比对照组显著增加(P<0.01),光照后CD54的表达反而略有下降。在光照和避光条件下,皮肤刺激剂、阴性受试物(乳酸)均未引起细胞表达CD54或CD86的显著性变化。以CD54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85.2%、100%和78.9%,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表面标志物评价指标为CD54,判定标准为:(1)光照后THP-1细胞表达CD54的MFI较照射前显著性增加;(2)光照后THP-1细胞表达CD54的RFI≥1.5;(3)当上述条件均满足时,对受试物进行预照射处理,结果仍然满足前2条标准。

Objective To identify and validate an in vitro photoallergy evaluation method with cell surface markers as research indicators.Methods THP-1 cells were incubated with a variety of photoallergy agents,photoirritants,skin sensitizers,skin irritant and negative subject respectively.After solar radiation or non-radiation treatment,the expression changes of cell surface markers CD54 and CD86 were detected by flow cytometry and analysed to confirm the specific evaluation indicators and to validate the accuracy,specificity,sensitivity and repeatability of the method.Results Among the 19 photoallergy agents,15 induced a significant increase in the mean fluorescence intensity(MFI)of CD54 expressed in THP-1 cells in the irradiated groups compared with the nonirradiated groups(P<0.05 and 0.01),and the relative fluorescence intensity(RFI)of CD54 in THP-1 cells in the irradiated groups were all above 1.5.The photoirritants also induced the significant increase in CD54 MFI of THP-1 cells after solar irradiation(P<0.01),but the expression level of CD54 was significantly decreased after pre-irradiation treatment compared with direct irradiation group(P<0.01).Without solar radiation,the expression levels of CD54 and CD86 in THP-1 cells incubated with the two skin sensitizers were significantly increased compared with control group(P<0.01),while the expression of CD54 was slightly decreased after irradiation.Both skin irritants and negative subject did not cause the significant change in CD54 and CD86 expression under solar irradiation and in the dark conditions.In this study,the accuracy,specificity and sensitivity of the photoallergy evaluation method using THP-1 cells with CD54 as the evaluation index was 85.2%,100%and 78.9%respectively,and the method had good repeatability.Conclusion The cell surface marker evaluation index of THP-1 cell photoallergy evaluation method was determined as CD54,and the evaluation criteria were as follows:(1)MFI of THP-1 cells expressing CD54 increased significantly after irradiation compared with before irradiation,(2)The RFI of CD54 expression in THP-1 cells after irradiation was≥1.5,(3)When all the above conditions are met,the test materials are pre-irradiated,and the results still meet the first two standards.