以细胞因子为研究指标的光致敏体外评价方法的建立

Establishment of an in vitro photosensitization evaluation method using cytokinesas evaluation index

目的研究和建立以细胞因子水平变化作为评价指标的光致敏体外评价方法。方法THP-1细胞分别与光致敏剂6-甲基香豆素(6-MC)和对氨基苯甲酸(PABA)、光致敏剂兼光刺激剂硫氯酚(bithionol)、光致敏兼皮肤致敏剂对苯二胺(PPD)和二苯甲酮(BP)、单纯皮肤致敏剂盐酸双氯苯双胍己烷(CHD)和二硝基氯苯(DNCB)、光刺激剂蒽(anthracene)和吖啶(acridine)、皮肤刺激剂月桂基磺酸钠(SDS)、阴性对照乳酸(LA)孵育24 h,与相应溶剂孵育作为对照组,经光照射(1.7 mW·cm^(−2),50 min)或避光处理后,细胞换液放入培养箱内继续培养5 h。光刺激剂需在正式光照射前进行预照射(在SOL 500人工太阳照射装置中照射30 min,然后避光放置15 min)处理。采用Luminex分析仪及多因子检测试剂盒分别检测细胞因子白细胞介素(IL)-12/P40、IL-12/P70、IL-1β、肿瘤坏死因子-α(TNF-α)、IL-8、IL-18、IL-4和IL-13水平的改变,确定光致敏相关特异性细胞因子,建立相应的光致敏体外评价方法。结果光致敏剂与THP-1细胞孵育并经光照后,与未照射组比较,细胞裂解液+上清液中IL-8和TNF-α的含量均显著增加(P<0.01),其中IL-8的增加幅度最大,光致敏剂6-MC、PABA、PPD和硫氯酚照射与未照射组的IL-8水平比值均大于10,TNF-α水平比值均大于1.5。光刺激剂吖啶和蒽细胞模型经过光直接照射后,IL-8水平是未照射组的10倍以上,TNF-α水平是未照射组的1.5倍以上;经过预照射处理后,其照射组与未照射组IL-8比值均下降到10以下,TNF-α比值均下降到1.5以下。皮肤致敏剂DNCB和CHD、皮肤刺激剂SDS、阴性对照LA和对照组,光照射与未照射组IL-8比值均小于10,TNF-α比值均小于1.5。结论在THP-1细胞模型中,IL-8和TNF-α对于评价化合物光致敏性具有较好的特异性和灵敏度,IL-8更优。

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-αin 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-αlevel in irradiation and non-irradiation groups were all greater than 1.5.The levels of IL-8 and TNF-αin 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-αratio 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-αratio 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-αhave good specificity and sensitivity for evaluating the photosensitivity of compounds,and IL-8 is better.