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

与衰老相关的基底膜胶原蛋白损失如何调节表皮屏障稳态？

• 批准号: 2547968
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2547968
• 关键词: How; does; ageing; related; loss

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.

AIM 1建立3D皮肤等效物，并结合了与Col7或Col17的shRNA敲低的永生化角质形成细胞。将使用免疫荧光检查整联蛋白受体，肌动蛋白，核细胞骨架和分化蛋白。将挖掘出具有Col7和Col17的敲低（KD）的角质形成细胞的现有RNASEQ数据，以指导进一步的分析。皮肤屏障将使用染料排除，尼罗河红色染色和转谷氨酰胺酶活性测定法进行表征。实验将使用带有Col7/Col17的KD的主要（1O）角质形成细胞复制。 AIM 2将机械/紫外线应力应用于AIM 1 3D皮肤当量。学生将在可拉伸的多孔膜（Gautrot Lab）上开发3D模型以应用机械伸展。另一种压力源是紫外线照射。同时，将使用Seahorse技术在具有Col7/Col17 KD的单层培养物中使用海马技术来测量糖酵解和氧化磷酸化。免疫染色将用于氧化应激，增殖和DNA损伤标记。通过对组蛋白标记的免疫染色和高分辨率共聚焦显微镜的免疫染色，将分析核形态和染色质重塑的变化。IAM3使用表观基因组方法来鉴定BM损失中鉴定出的转录反应的调节剂。 AIM 2的角质形成细胞将使用选定的转录调节剂进行抗体进行染色质免疫沉淀（CHIP），然后进行下一代测序（NGS）。学生将将这些数据与现有的RNASEQ数据集成在一起，以确定BM如何调节表皮稳态。可以通过免疫染色来比较年轻和老化的皮肤的发现。IAM4鉴定和测试化合物可减轻BM改变对表皮稳态联合抗体，生物信息学和化学 - 信息性工具的影响，以通过基于RNA的签名分析，路径驱动器和目标/铅/引导ID方法来识别化合物。原理选项的证明将在老年角质形成细胞模型中进行测试，以检查是否可以改善屏障和地下膜。