目的 研究和建立以細胞因子水平變化作為評價指標的光致敏體外評價方法。方法 THP-1細胞分別與光致敏劑6-甲基香豆素（6-MC）和對氨基苯甲酸（PABA）、光致敏劑兼光刺激劑硫氯酚（bithionol）、光致敏兼皮膚致敏劑對苯二胺（PPD）和二苯甲酮（BP）、單純皮膚致敏劑鹽酸雙氯苯雙胍己烷（CHD）和二硝基氯苯（DNCB）、光刺激劑蒽（anthracene）和吖啶（acridine）、皮膚刺激劑月桂基磺酸鈉（SDS）、陰性對照乳酸（LA）孵育24 h，與相應(yīng)溶劑孵育作為對照組，經(jīng)光照射（1.7 mW·cm^-2，50 min）或避光處理后，細胞換液放入培養(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行預照射（在SOL 500人工太陽照射裝置中照射30 min，然后避光放置15 min）處理。采用Luminex分析儀及多因子檢測試劑盒分別檢測細胞因子白細胞介素（IL）-12/P40、IL-12/P70、IL-1β、腫瘤壞死因子-α（TNF-α）、IL-8、IL-18、IL-4和IL-13水平的改變，確定光致敏相關(guān)特異性細胞因子，建立相應(yīng)的光致敏體外評價方法。結(jié)果 光致敏劑與THP-1細胞孵育并經(jīng)光照后，與未照射組比較，細胞裂解液＋上清液中IL-8和TNF-α的含量均顯著增加（P<0.01） ，其中IL-8的增加幅度最大，光致敏劑6-MC、PABA、PPD和硫氯酚照射與未照射組的IL-8水平比值均大于10，TNF-α水平比值均大于1.5。光刺激劑吖啶和蒽細胞模型經(jīng)過光直接照射后，IL-8水平是未照射組的10倍以上，TNF-α水平是未照射組的1.5倍以上；經(jīng)過預照射處理后，其照射組與未照射組IL-8比值均下降到10以下，TNF-α比值均下降到1.5以下。皮膚致敏劑DNCB和CHD、皮膚刺激劑SDS、陰性對照LA和對照組，光照射與未照射組IL-8比值均小于10，TNF-α比值均小于1.5。結(jié)論 在THP-1細胞模型中，IL-8和TNF-α對于評價化合物光致敏性具有較好的特異性和靈敏度，IL-8更優(yōu)。

Objective To establish an in vitro photosensitization evaluation method using cytokines as evaluation index. Methods THP-1 cells were treated with photosensitizers 6-methylcoumarin (6-MC) and P-aminobenzoic acid (PABA), photosensitizers and photostimulants Bithionol, photosensitizers and skin sensitizers p-phenylenediamine (PPD) and diphenylketone (BP), and pure skin sensitizers dichlorobenidine hydrochloride (CHD) and dinitrochlorobenzene (DNCB), photostimulant anthracene and acridine, skin irritant sodium lauryl sulfonate (SDS), and negative control lactic acid (LA) were incubated for 24 h, and the corresponding solvent group was used as the control group. After light irradiation (1.7 mW·cm^-2, 50 min) or light protection treatment, cells were placed into the incubator for further culture for 5 h. Photostimulant should be pre-irradiated before formal light exposure (30 min in SOL 500 artificial solar irradiation device, and then placed in dark for 15 min). Luminex analyzer and multifactor detection kit were used to detect the changes of cytokines interleukin (IL)-12/P40, IL-12/P70, IL-1β, tumor necrosis factor-α (TNF-α), IL-8, IL-18, IL-4 and IL-13, respectively, to determine the photosensitization related specific cytokines. To establish an in vitro evaluation method for photosensitization. Results After incubation with the photosensitizers and exposure to light, the contents of IL-8 and TNF-α in the cell lysate and supernatant of THP-1 cells were significantly increased (P < 0.01), with the largest increase magnitude in IL-8. For the four photosensitizers, the ratios of IL-8 level in irradiation and non-irradiation groups were all greater than 10, and the ratios of TNF- α level in irradiation and non-irradiation groups were all greater than 1.5. The levels of IL-8 and TNF- α in acridine and anthracene cell models exposed to light were more than 10 times and 1.5 times higher than those in the unirradiated group. After preirradiation, the IL-8 ratio of the irradiated group and the unirradiated group decreased to less than 10, and the TNF-α ratio decreased to less than 1.5. Skin sensitizers DNCB and CHD, skin irritant SDS, negative control LA and control group, IL-8 ratio was less than 10 and TNF-α ratio was less than 1.5 in light irradiation and non-irradiation groups. Skin irritants and negative test substances did not cause significant changes in the levels of these two cytokines. Conclusion In the THP-1 cell model, IL-8 and TNF-α have good specificity and sensitivity for evaluating the photosensitivity of compounds, and IL-8 is better.

R965.1

國家“重大新藥創(chuàng)制”科技重大專項（2018ZX09201017）