Screening and identification of pericyte-specific and subpopulation-specific markers

项目摘要/摘要 这项应用的长期目标是充分了解周细胞的生物学功能。 生理和病理条件，并开发以周细胞为基础的创新疗法 与衰老相关的疾病，这与NIA的使命一致。这项提议旨在解决一个 周细胞研究领域的关键问题/障碍：缺乏特定亚群和年龄- 特定的/与年龄无关的周细胞标志物。我们建议筛选和鉴定分子标记 使用一种独特的转基因/生化方法对大脑和肌肉周细胞进行特异性检测，随后 RNAseq分析和随后的创新筛选/比较战略。在目标1中，周细胞和 血管平滑肌细胞(VSMCs)将从脑组织和骨骼肌中分离出来 AI14：SM22α-Cre小鼠使用我们实验室优化的基于流式细胞仪的方案。由于vSMC- Td番茄在这个转基因品系中的特异性表达，这种方法与其他方法不同，允许分离 周细胞和血管平滑肌细胞。下一步，新分离的周细胞和vSMC将受到RNAseq的影响 分析。周细胞的转录图谱将首先相对于VSMC的转录图谱进行负选择， 以及富含周细胞的基因将进一步与其他细胞的转录本进行比较(可从 公共数据库)。周细胞特有的基因是潜在的周细胞特异性标记。通过比较 来自幼年和老年小鼠的周细胞特异性基因、年龄特定和年龄无关的标记将是 已确认身份。通过比较大脑和肌肉周细胞特有的基因，亚群特异性标记 将会被确认。在目标2中，RNAseq确定的候选基因将在体外和体内得到验证，以及 验证的细胞表面标记在基于FACS的周细胞分离中的应用将被评估。 该项目的成功完成不仅将识别周细胞特异性标记，还将识别 亚群特异性(脑与肌肉)和年龄特定/年龄无关(年轻与老年)周细胞 记号笔。这些标记将能够以高纯度分离活的周细胞，用于体外研究，靶向 在体内专门用于功能丧失研究的周细胞，以及在特定亚型中研究周细胞 以及特定于年龄/独立于年龄的方式。这些研究将极大地丰富我们在 周细胞生物学/功能，产生新的遗传工具，为新的研究方向打开大门，以及 大大推动了这一领域的发展。

Project Summary/Abstract The long-term objective of this application is to fully understand the biological functions of pericytes in physiological & pathological conditions and develop pericyte-based innovative therapies for various aging-related disorders, which is consistent with the mission of NIA. This proposal aims to solve a critical problem/barrier in the field of pericyte research: the lack of subpopulation-specific and age- specific/age-independent pericyte markers. We propose to screen and identify molecular markers specific for brain and muscle pericytes using a unique transgenic/biochemical approach, followed by RNAseq analyses and subsequent innovative screening/comparing strategy. In Aim 1, pericytes and vascular smooth muscle cells (vSMCs) will be isolated from the brains and skeletal muscles of Ai14:SM22α-Cre mice using FACS-based protocols optimized in our laboratory. Due to the vSMC- specific expression of tdTomato in this transgenic line, this approach, unlike others, allows separation of pericytes and vSMCs. Next, freshly isolated pericytes and vSMCs will be subjected to RNAseq analyses. The transcriptional profile of pericytes will be first negatively selected against that of vSMCs, and pericyte-enriched genes will be further compared with transcriptomes of other cells (available from public databases). Genes unique to pericytes are potential pericyte-specific markers. By comparing pericyte-specific genes from young and old mice, age-specific and age-independent markers will be identified. By comparing genes unique to brain and muscle pericytes, subpopulation-specific markers will be identified. In Aim 2, RNAseq-identified candidate genes will be validated in vitro and in vivo, and the application of validated cell-surface markers in FACS-based pericyte isolation will be assessed. Successful completion of this project will not only identify pericyte-specific markers, it will also identify subpopulation-specific (brain vs. muscle) and age-specific/age-independent (young vs. old) pericyte markers. These markers will enable isolating live pericytes at high purity for in vitro studies, targeting pericytes specifically in vivo for loss-of-function studies, and studying pericytes in a subtype-specific and age-specific/age-independent manner. These studies will dramatically enrich our knowledge in pericyte biology/function, generate new genetic tools, open doors for new lines of research, and substantially move the field forward.