The effects of UVB and the organic osmolyte betaine on rat epidermal keratinocytes in an organotypic culture system. Rattus norvegicus

Betaine (trimethylglycine) is a non-toxic, highly water-soluble organic osmolyte widely used in skin care due to its assumed moisturizing and protective properties, but only few studies have addressed its specific effects in skin. Here, the cellular and molecular targets of betaine were analyzed by genome-wide expression analysis in organotypic cultures of rat epidermal keratinocytes (REK). In this model, we also examined whether betaine modifies the impacts of acute UVB exposure. The expression of several genes relevant to epidermal biology, proliferation/differentiation or malignancy as well as solute transport were verified by independent methods (qRT-PCR, western blotting). The data concerning changes in calcium metabolism after UVB exposure has been published separately (Bart et al., Br. J. Dermatol. 171:376-387, 2014). Overall design: Organotypic cultures of rat epidermal keratinocytes (REK) were prepared by plating cells on a collagen-coated insert (submerged in medium), lifting the confluent cell layer to the air-liquid interface 3 days after plating. These 3D cultures were divided in four treatment groups with three replicates in each: control, betaine (10 mM), UVB (30 mJ/cm2) and betaine + UVB. Betaine was added to the cultures for 11 days, starting from day 4 until sample collection. UVB exposure was performed 24 h prior to sample collection for 2-week-old 3D cultures with a well-formed epidermal layer. For further details on the experimental set-up and characteristics of the UV source, please refer to Bart et al. (Br. J. Dermatol. 171:376-387, 2014) and Rauhala et al. (J. Biol. Chem. 288:17999-18012, 2013).

甜菜碱（Betaine，三甲基甘氨酸）是一种无毒、高水溶性的有机渗透调节物质，因其兼具保湿与防护功效而被广泛应用于护肤品中，但目前针对其在皮肤中具体作用的研究寥寥无几。本研究通过全基因组表达分析，对大鼠表皮角质形成细胞（rat epidermal keratinocytes, REK）的器官型培养体系中甜菜碱的细胞与分子靶点进行了探究。在此模型中，我们同时考察了甜菜碱是否会改变急性紫外线B（UVB）暴露所产生的影响。与表皮生物学、增殖/分化或恶性肿瘤相关的多种基因以及溶质转运相关基因的表达变化，均通过独立实验方法（qRT-PCR、蛋白质印迹法）得到了验证。有关UVB暴露后钙代谢变化的相关数据已另行发表（Bart等，Br. J. Dermatol. 171:376-387, 2014）。总体实验设计如下：将大鼠表皮角质形成细胞（REK）接种于包被胶原蛋白的插入式培养板（浸没于培养基中），待细胞汇合后于接种第3天将其转移至气液界面进行培养。将这些三维培养体系分为4个处理组，每组设置3个生物学重复：对照组、甜菜碱组（10 mM）、UVB组（30 mJ/cm²）以及甜菜碱+UVB联合处理组。甜菜碱自接种第4天起添加至培养基中，持续培养11天直至样本收集。对于培养至2周、表皮层结构完整的三维培养体系，我们在样本收集前24小时进行UVB暴露处理。若需了解实验设置及紫外光源相关参数的更多细节，请参阅Bart等（Br. J. Dermatol. 171:376-387, 2014）及Rauhala等（J. Biol. Chem. 288:17999-18012, 2013）的研究成果。