[關(guān)鍵詞]
[摘要]
目的 確定并驗證以細胞表面標志物CD54作為評價指標的光致敏體外評價方法。方法 將THP-1細胞與多種光致敏劑、光刺激劑、皮膚致敏劑、皮膚刺激劑和陰性受試物分別孵育,在光照射或避光處理后,用流式細胞儀測定細胞表面標志物CD54和CD86的表達水平并統(tǒng)計分析,進一步確定具體的評價指標,并對該方法的準確性、特異性、靈敏度、重復性進行驗證。結(jié)果 19種光致敏劑中有15種引起照射組THP-1細胞表達CD54的平均熒光強度(MFI)較非照射組顯著增加(P<0.05、0.01) ,且照射組細胞表達CD54的相對熒光強度(RFI)值均在1.5以上。光刺激劑經(jīng)光照射后也可引起細胞表達CD54的MFI顯著增加(P<0.01) ,但經(jīng)過預照射處理后,CD54的表達水平較直接照射組顯著下降(P<0.01)。未經(jīng)光照射條件下,皮膚致敏劑即可引起THP-1細胞表達CD54和CD86的MFI比對照組顯著增加(P<0.01) ,光照后CD54的表達反而略有下降。在光照和避光條件下,皮膚刺激劑、陰性受試物(乳酸)均未引起細胞表達CD54或CD86的顯著性變化。以CD54為評價指標的THP-1細胞光致敏評價方法檢測光致敏劑的準確性、特異性和靈敏度分別是85.2%、100%和78.9%,具有良好的重復性。結(jié)論 確定了THP-1細胞光致敏評價方法的細胞表面標志物評價指標為CD54,判定標準為:(1)光照后THP-1細胞表達CD54的MFI較照射前顯著性增加;(2)光照后THP-1細胞表達CD54的RFI≥1.5;(3)當上述條件均滿足時,對受試物進行預照射處理,結(jié)果仍然滿足前2條標準。
[Key word]
[Abstract]
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.
[中圖分類號]
R965.1
[基金項目]
國家“重大新藥創(chuàng)制”科技重大專項資助項目“創(chuàng)新藥物非臨床安全性評價研究關(guān)鍵技術(shù)”(2018ZX09201017)