目的 確定并驗(yàn)證以白細(xì)胞介素-8 （IL-8）作為評(píng)價(jià)指標(biāo)的THP-1細(xì)胞光致敏體外評(píng)價(jià)方法。方法 將THP-1細(xì)胞與19種光致敏劑、4種光刺激劑、2種皮膚致敏劑、1種皮膚刺激劑和陰性受試物分別孵育24 h，在光照射（1.7 mW·cm^-2，50 min）或避光處理后，細(xì)胞換液放入培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)5 h。光刺激劑需在正式光照射前進(jìn)行預(yù)照射（光照30 min，然后避光15 min）處理。用Luminex液相芯片檢測(cè)技術(shù)測(cè)定細(xì)胞培養(yǎng)上清和細(xì)胞裂解液中IL-8和腫瘤壞死因子-α （TNF-α）的含量并統(tǒng)計(jì)分析，進(jìn)一步確定評(píng)價(jià)光致敏的細(xì)胞因子指標(biāo)，并進(jìn)行方法驗(yàn)證。結(jié)果 與非照射組比較，13種光致敏劑均引起照射組THP-1細(xì)胞IL-8總含量顯著增加（P<0.01） ，阿伏苯宗為非照射組的1 148～2 269倍，其余12種為非照射組的6.7～195.1倍；光刺激劑經(jīng)光照射后也可引起細(xì)胞分泌IL-8顯著增加（P<0.01） ，但經(jīng)過(guò)預(yù)照射處理后，IL-8的含量相比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(xì)胞分泌IL-8比對(duì)照組顯著增加（P<0.01） ，光照后IL-8含量雖也較非照射組顯著增加（P<0.01） ，但增加的幅度小于皮膚致敏劑本身引起IL-8變化的幅度。在光照條件下，皮膚刺激劑、陰性受試物均可引起細(xì)胞分泌IL-8較非照射組顯著性增加（P<0.05） ，但增加的幅度較小，與對(duì)照組相似。以上受試物引起光照前后細(xì)胞TNF-α變化的幅度均與對(duì)照組相近。以IL-8為評(píng)價(jià)指標(biāo)的THP-1細(xì)胞光致敏評(píng)價(jià)方法檢測(cè)光致敏劑的準(zhǔn)確性、特異性和靈敏度分別是77.8%、100.0%、68.4%，并且該方法具有良好的重復(fù)性。結(jié)論 確定了THP-1細(xì)胞光致敏評(píng)價(jià)方法的細(xì)胞因子標(biāo)志物評(píng)價(jià)指標(biāo)為IL-8，判定標(biāo)準(zhǔn)為： ①UVA照射后THP-1細(xì)胞中IL-8含量比非照射組顯著增加；②UVA照射組與非照射組IL-8含量比值≥6.5；③當(dāng)上述兩條均滿足時(shí)，對(duì)受試物進(jìn)行UVA預(yù)照射處理，預(yù)處理照射組IL-8的含量與直接照射組（未經(jīng)預(yù)照射）相比無(wú)顯著性差異，且預(yù)處理照射組與預(yù)處理非照射組IL-8含量比值≥6.5。

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 1 148 to 2 269 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-α level between irradiation and non irradiation groups of the above test articles were similar to that of the cell control. In this study, the accuracy, specificity and sensitivity of the photoallergy evaluation method using THP-1 cells with IL-8 as the evaluation index was 77.8%, 100.0% and 68.4%, respectively, and the method had good repeatability. Conclusion In this study, a photoallergy evaluation method using THP-1 cells with IL-8 as the evaluation index was further determined and was verified to have good accuracy, specificity, sensitivity and repeatability.

R965.1

國(guó)家“重大新藥創(chuàng)制”科技重大專(zhuān)項(xiàng)資助項(xiàng)目“創(chuàng)新藥物非臨床安全性評(píng)價(jià)研究關(guān)鍵技術(shù)”（2018ZX09201017）