How does ageing-related loss of basement membrane collagen regulate epidermal barrier homeostasis?

目的1建立含有Col7或Col17 shRNA敲除的永生化角质形成细胞的3D皮肤等效物。整合素受体、肌动蛋白、核细胞骨架和分化蛋白将使用免疫荧光进行检测。来自角质形成细胞的现有的具有Col7和Col17基因敲除(Kd)的RNAseq数据将被挖掘以指导进一步的分析。皮肤屏障将通过染料排斥、尼罗河红染色和转谷氨酰胺酶活性测定来表征。实验将使用具有Col7/Col17 Kd的原代(10)角质形成细胞进行复制。目的2将机械/UV应力施加到目标1的3D皮肤等效物上。学生将在可拉伸的多孔膜(Gautrot实验室)上开发3D模型，以应用机械拉伸。另一种应激源是紫外线照射。同时，糖酵解和氧化磷酸化将在含有Col7/Col17的Kd的单层培养中使用SeaHorse技术进行测量。将进行氧化应激、增殖和DNA损伤标记物的免疫染色。核形态和染色质重塑的变化将通过组蛋白标记的免疫染色和高分辨率共聚焦显微镜进行分析。Aim 3使用表观基因组学方法识别BM丢失所识别的转录反应的调节因子。来自AIM 2的角质形成细胞将接受染色质免疫沉淀(ChIP)，使用选定的转录调节因子抗体，然后进行下一代测序(NGS)。学生将把这些数据与现有的RNAseq数据相结合，以确定BM是如何调节表皮动态平衡的。通过免疫染色可以比较年轻和老年皮肤的研究结果。目标4识别和测试化合物以减轻BM改变对表皮自身稳定性的影响在联合利华，生物信息学和化学信息学工具将通过基于RNA的签名分析、途径驱动和目标/先导ID方法的组合来识别化合物。原则证明选项将在老化角质形成细胞模型中进行测试，以检查屏障和基底膜是否可以改善。

Aim 1 Establish 3D skin equivalents incorporating immortalised keratinocytes with shRNA knockdown of Col7 or Col17. Integrin receptors, actin, nucleocytoskeleton and differentiation proteins will be examined using immunofluorescence. Existing RNAseq data from keratinocytes with knockdown (KD) of Col7 and Col17 will be mined to direct further analyses. The skin barrier will be characterized using dye exclusion, Nile Red staining and transglutaminase activity assays. Experiments will be replicated using primary (1o) keratinocytes with KD of Col7/Col17. Aim 2 Apply mechanical/UV stress to the Aim 1 3D skin equivalents. The student will develop the 3D models on a stretchable porous membrane (Gautrot lab) to apply mechanical stretch. An alternative stressor is UV irradiation. In parallel, glycolysis and oxidative phosphorylation will be measured using Seahorse technology in monolayer cultures with KD of Col7/Col17 . Immunostaining will be performed for oxidative stress, proliferation and DNA damage markers. Changes in nuclear morphology and chromatin remodelling will be analysed by immunostaining of histone marks and high-resolution confocal microscopy.Aim 3 Use epigenomic methods to identify regulators of the identified transcriptional response from BM loss. Keratinocytes from Aim 2 will be subjected to chromatin immunoprecipitation (ChIP) using antibodies for selected transcriptional regulators followed by Next Generation Sequencing (NGS). The student will integrate these data with the existing RNAseq data to identify how the BM is regulating epidermal homeostasis. Findings can be compared in young and aged skin by immunostaining.Aim 4 Identify and test compounds to mitigate effects of BM alterations on epidermal homeostasisAt Unilever, bioinformatic and chemi-informatic tools will be used to identify compounds via a combination of RNA-based signature analysis, pathway-driven and target/lead ID approaches. Proof of principle options will be tested in aged keratinocyte models to examine if the barrier and basement membrane can be improved.