Yasui Hiroyuki, Hakozaki Tomohiro, Date Akira, Yoshii Takashi, Sakurai Hiromu
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Japan.
Biochem Biophys Res Commun. 2006 Aug 18;347(1):83-8. doi: 10.1016/j.bbrc.2006.06.046. Epub 2006 Jun 16.
The objective of this study is to image and detect reactive oxygen species (ROS) generated in the UVB-exposed three-dimensional human skin equivalent model (HSEM), EpiDermtrade mark 200, because the alternative system needs to be urgently established as a replacement for the skin of experimental animals. Evidence that the ROS generation is enhanced in the skin of live animals after the UVB exposure was already obtained, by using the real-time chemiluminescent (RT-CL) method consisting of a sensitive CL probe (CLA) and an ultra-low light imaging apparatus. In this study, CL emission due to the reaction of CLA with endogenously generated ROS increased significantly in the UVB-treated HSEM compared with that in the intact HSEM, the maximum level being observed at a dose of 27mJ/cm(2). ROS under UVB exposure was identified to be ()O2- and (1)O(2) as observed by suppressive effects of SOD and beta-carotene topically applied on sample surface before the UVB exposure. The results for UVB-induced ROS generation in HSEM were consistent with those observed in the skin of live animals. HSEM combined with the RT-CL method was shown to be useful system not only to predict UVB-induced ROS-related skin responses in human but also to find protective agents against UVB-stimulated oxidative stress in place of animals and ex vivo human skin.
本研究的目的是对在紫外线B（UVB）照射的三维人体皮肤等效模型（HSEM）EpiDerm™ 200中产生的活性氧（ROS）进行成像和检测，因为迫切需要建立替代系统来取代实验动物的皮肤。通过使用由灵敏的化学发光（CL）探针（CLA）和超弱光成像设备组成的实时化学发光（RT-CL）方法，已经获得了UVB照射后活体动物皮肤中ROS生成增加的证据。在本研究中，与完整的HSEM相比，UVB处理的HSEM中由于CLA与内源性生成的ROS反应而产生的CL发射显著增加，在27mJ/cm² 的剂量下观察到最大水平。通过在UVB照射前局部应用于样品表面的超氧化物歧化酶（SOD）和β-胡萝卜素的抑制作用观察到，UVB照射下的ROS被鉴定为超氧阴离子（·O₂⁻）和单线态氧（¹O₂）。HSEM中UVB诱导的ROS生成结果与在活体动物皮肤中观察到的结果一致。结果表明，HSEM与RT-CL方法相结合不仅是预测人类UVB诱导的ROS相关皮肤反应的有用系统，而且是寻找替代动物和离体人类皮肤的抗UVB刺激氧化应激保护剂的有用系统。
