紫外线A诱导的人真皮成纤维细胞光老化及小鼠皮肤光老化模型的构建与评价 ^*

Construction and evaluation of photoaging models of human dermal fibroblasts and mouse skin induced by ultraviolet A

摘要

目的 建立紫外线A (UVA) 诱导的人真皮成纤维细胞 (HDFs) 光老化和小鼠皮肤光老化模型, 为探究皮肤光老化机制及防治皮肤光老化提供良好的模型。方法 利用酶消化法分离获得HDFs, 并检测其表面标志物。采用UVA照射HDFs (剂量5 J/cm ², 15 min/d, 连续3 d) 构建体外HDFs光老化模型。将12只雌性昆明小鼠分为对照组 (Control组) 和模型组 (UVA照射组), 每组6只。采用UVA照射小鼠 (剂量20 J/cm ², 1 h/d, 5次/周, 连续8周), 构建小鼠皮肤光老化模型。通过观察细胞形态、检测其增殖能力和衰老相关β-半乳糖苷酶活性, 小鼠背部皮肤的大体观察, 皮肤组织苏木素-伊红和Masson染色, 实时荧光定量聚合酶链反应 (RT-qPCR) 检测衰老相关因子P16 ^INK4a和P21的mRNA表达, 以及Western blot和免疫组织化学染色检测皮肤老化相关蛋白Ⅰ型胶原蛋白 (Collagen-Ⅰ) 和基质金属蛋白酶1 (MMP1) 的表达, 对模型进行评价。结果 HDFs呈长梭形, 表达波形蛋白而不表达E钙黏蛋白。UVA照射后HDFs体积变大、呈铺展状, 其增殖能力减弱, 衰老细胞阳性率及P16 ^INK4a和P21的mRNA表达水平增高, Collagen-Ⅰ表达下调, 而MMP1表达上调。与Control组比较, UVA照射组小鼠的背部皮肤粗糙, 可见色素沉着, 其表皮增厚, 真皮变薄, 胶原沉积减少, 胶原纤维排列紊乱, 且P16 ^INK4a和P21的mRNA表达水平显著增高, Collagen-Ⅰ表达降低, 而MMP1表达升高。结论 成功建立了UVA照射所致的HDFs光老化模型和小鼠皮肤光老化模型, 为探究皮肤光老化的机制以及开发光老化防治策略提供了实验基础。

Abstract

Objective To establish photoaging models of human dermal fibroblasts (HDFs) and mouse skin induced by ultraviolet A (UVA), so as to provide the optimized models for exploring mechanisms of skin photoaging and preventing and treating skin photoaging. Methods Enzyme digestion was used to isolate HDFs and surface markers were examined. UVA at a dose of 5 J/cm ² was utilized to irradiate HDFs for 15 minutes each day for three consecutive days, and then in vitro HDF photoaging models were established. Twelve female Kunming mice were divided into a control group and a model group (UVA irradiation group) with six mice in each group. A dose of 20 J/cm ² of UVA was applied for 1 hour daily, 5 times a week, which was continued for eight weeks to establish a mouse skin photoaging model. Furthermore, the model efficacies were assessed by observing the cellular morphology, detecting the cell proliferation ability and the activity of senescence-related β-galactosidase, observing the gross appearance of the dorsal skin of mice, performing hematoxylin-eosin staining and Masson staining of the skin tissues, detecting the expression of P16 ^INK4a and P21 mRNA via RT-qPCR, and examining the expression of type Ⅰ collagen (Collagen-Ⅰ) and matrix metalloproteinase 1 (MMP1) by Western blot and immunohistochemical staining. Results HDFs were long fusiform-shaped, which expressed vimentin but did not express E-cadherin. After UVA irradiation, the size of HDFs became larger with spreading morphology, along with attenuated proliferation ability. And the positive rate of senescent cells and the expression levels of P16 ^INK4a and P21 mRNA were up-regulated. Meanwhile, Collagen-Ⅰ expression was declined, while MMP1 expression was elevated. Compared with the control group, the dorsal skin of mice was rough with pigmentation after UVA irradiation. UVA exposure gave rise to thickening of the epidermis and thinning of the dermis with decreased and disorganized collagen. Moreover, the expression levels of P16 ^INK4a and P21 mRNA in UVA irradiation group were dramatically higher than those in the control group. And the expression of Collagen-Ⅰ diminished, while the expression of MMP1 enhanced. Conclusion The UVA-induced HDF photoaging model and mouse skin photoaging model are successfully established, which provide an experimental basis for exploring the mechanism of skin photoaging and developing therapeutic strategies.

关键词

皮肤光老化 / 紫外线A / 成纤维细胞 / 动物模型 / 评价

Key words

Skin photoaging / Ultraviolet A / Fibroblasts / Animal model / Evaluation

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基金资助

^*吉林省科技发展计划项目(20240404038ZP)