体外银屑病样三维皮肤模型的构建_解雨馨.pdf

基金项目四川省科技厅项目(2021YJ0233)作者单位西南医科大学附属医院皮肤科，四川 泸州 646000通信作者钟建桥，E-mail:2842023 年 3 月第 37 卷第 3 期Mar.2023，Vol37，No3http:/体外银屑病样三维皮肤模型的构建解雨馨，彭慧玲，先德海，钟建桥 摘要目的通过应用肿瘤坏死因子-(tumor necrosis factor-alpha，TNF-)和-干扰素(interferon-gamma，IFN-)联合诱导建立体外银屑病样三维皮肤模型，为银屑病发病机制及其治疗的研究提供理想的实验载体。方法从 2 10 岁男童包皮组织中分离出原代角质形成细胞(keratinocytes，KCs)和成纤维细胞(fibroblast，FBs)进行贴壁培养。常规传至 2 3 代后，FBs 与型鼠尾胶原混合获得真皮类似物，然后将 KCs 接种到 Transwell 小室行气-液培养构建出正常三维皮肤模型。在正常三维皮肤模型气-液培养的最后4 d，每天在上室各加入 7.5 ng/mL 的 TNF-和 IFN-以构建银屑病样三维皮肤模型。采用 HE 染色(hematoxylin-eosin staining，HE)观察模型组织病理学变化，免疫组织化学染色检测 KCs 的 K17 和 Ki67 表达，ELISA 测定模型上清液中 IL-17、IL-22、IL-23、OS、MDA、SOD、GSH 等水平。结果正常三维皮肤模型外观呈乳白色半透明状，边缘整齐;而银屑病样三维皮肤模型呈乳白色不透明状，边缘蜷曲，较正常皮肤模型明显增厚、缩小。组织病理学示，正常三维皮肤模型表皮层由 1 3 层整齐排列的 KCs 构成，真皮层见梭形 FBs和胶原;银屑病样三维皮肤模型表皮厚度显著增加，真皮层由梭形 FBs 和红色胶原组成。免疫组织化学染色示:银屑病样三维皮肤模型表皮层细胞胞浆 K17 呈阳性表达，约 70%胞核表达 Ki67 阳性;而正常三维皮肤模型表皮细胞几乎未见 K17表达，仅少量细胞 Ki67 阳性。银屑病样三维皮肤的 IL-17、IL-22、IL-23、OS 和MDA 水平较正常三维皮肤显著升高(P 0.01)，而 SOD 和 GSH 表达明显下降(P 0.01)。结论经 TNF-和 IFN-干预正常三维皮肤模型后，可成功构建出银屑病样三维皮肤模型。该模型具有与人银屑病相似的形态学和分子生物学特征，为进一步研究银屑病的发病机制和药物的研发提供了理想的实验载体。关键词 银屑病;三维皮肤模型;TNF-;IFN-中图分类号 75863 文献标志码 A 文章编号 1001 7089(2023)03 0284 07 DOI 10 13735/j cjdv1001-7089 202209006Study on Establishment of Psoriasis-Like Three-Dimensional Skin Model in VitroXIE Yuxin，PENG Huiling，XIAN Dehai，ZHONG Jianqiao(Department of Dermatology，the Affiliated Hospital of Southwest Medical University，Luzhou646000，China)Corresponding author ZHONG Jianqiao，E-mail: Abstract ObjectiveTo establish a psoriasis-like three-dimensional(3D)skin model in vitrothrough combined use of tumor necrosis factor-lpha(TNF-)with interferon-gamma(IFN-)，thus providing an ideal experimental carrier for the investigation of thepathogenesis and treatment of psoriasis MethodsPrimary keratinocytes(KCs)and解雨馨，彭慧玲，先德海，等 体外银屑病样三维皮肤模型的构建http:/fibroblasts(FBs)were respectively isolated and adherently cultured from the foreskintissues of boys aged 2-10 years.After routinely passaged for 2-3 times，FBs weremixed with type rat tail collagen to obtain a dermal analogue，and then KCs wereinoculated into Transwell chamber for air-liquid culture to construct a normal 3D skinmodel.In the last 4 days of air-liquid culture of normal 3D skin model，7.5 ng/mL ofTNF-and IFN-were added to the upper chamber respectively daily to establish thepsoriasis-like 3D skin model.The histopathological changes of psoriasis-like 3D skinmodel were observed by hematoxylin-eosin staining(HE)，and mmunohistochemicalstaining was used to detect the expression of K17 and Ki67 in KCs.ELISA wasperformed to determine the levels of IL-17，IL-22，IL-23，OS，MDA，SOD andGSH in the supernatant of the model esultsThe appearance of the normal 3D skinmodel was white and semitransparent with even edges，while the psoriasis-like 3Dskin model manifested as the milky white and opaque appearance with thickening andcurled edge，obviously smaller than the normal 3D skin model.In histopathology，theepidermis of normal 3D skin model was composed of 1-3-layer well-arranged KCs，with spindle FBs and collagen in the dermis，whereas the epidermis markedlythickened and the dermis still consisted of spindle FBs and red collagen in psoriasis-like 3D skin model.Immunohistochemical analysis revealed a positive expression ofK17 in the cytoplasm and positive Ki67 in 70%of the nuclei of the epidermal cells inpsoriasis-like 3D skin model，while K17 scarcely appeared in the epidermal cells ofthe normal 3D skin model，and positive Ki67 was seen in only few cells.Comparedwith the normal 3D skin，the protein levels of IL-17，IL-22，IL-23，OS and MDAsignificantly increased in the psoriasis-like 3D skin(P 0.01)，while SOD and GSHremarkably decreased(P 0.01)ConclusionThe psoriasis-like 3D skin model canbe successfully constructed through intervention of normal 3D skin model with TNF-and IFN-.It exhibits similar morphological and molecular biological characteristicsto human psoriasis，which offers an ideal experimental carrier for further study on thepathogenesis of psoriasis and drug development.Key words Psoriasis;Three-dimensional(3D)skin model;TNF-;IFN-银屑病是一种慢性炎症性疾病，与遗传易感性、免疫、炎症、氧化应激、外界环境等因素密切相关，影响全球人群约 2%3%1。在组织学上主要表现为角质形成细胞(keratinocytes，KCs)异常增殖、真皮毛细血管增生和炎性细胞浸润。目前银屑病的发病机制仍不完全清楚，但炎症和氧化应激被认为是致其发病的重要促进因素2-3。为更好地阐明银屑病的发病机制进而开发相应的药物，各种体内外模型得以快速发展。虽然动物模型能在一定程度上模拟人银屑病，但由于动物和人体的差异性与伦理学争议，使该类模型在发病机制研究和新药疗效观察方面仍受局限。基于此，体外模型得到了很好的发展，尤其是体外三维皮肤模型具有比单层细胞模型更符合人体皮肤结构和功能的特点4-5。故本研究通过联合应用肿瘤坏死因子-(tumor necrosis factor-alpha，TNF-)和-干扰素(interferon-gamma，IFN-)干预正常三维皮肤模型，建立具有类银屑病形态学、分子生物学特征的银屑病样三维皮肤模型，为进一步探究银屑病的发病机制以及后续药物研发提供理想的实验模型。1材料与方法1.1材料1.1.1实验细胞构建三维皮肤模型的细胞所需要的原代 KCs 和成纤维细胞(fibroblasts，FBs)，均来自西南医科大学附属医院行包皮环切术的 2 10岁健康儿童的包皮。该研究获得西南医科大学附属医院伦理委员会的批准(2015016A)，同时已获取患儿家属的书面同意，签署知情同意书。582http:/1.1.2主要试剂型鼠尾胶原蛋白购自北京鸿跃生物科技有限公司;TNF-和 IFN-购自美国 POTEINTECH GOUP 公司;白细胞介素 17(interleukin-17，IL-17)、IL-22、IL-23 及活性氧(reactiveoxygen species，OS)试剂盒购自北京安迪华泰科技有限公司;丙二醛(malondialdehyde，MDA)、超氧化物歧化酶(superoxide dismutase，SOD)及谷胱甘肽(glutathione，GSH)试剂盒购自南京建成科技有限公司;角蛋白 17(keratin-17，K17)、Ki67 抗体购自Abcam 公司。1.2方法1.2.1细胞