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

周细胞特异性和亚群特异性标记物的筛选和鉴定

基本信息

批准号：
10609234
负责人：
Yao Yao
金额：
$ 18.87万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-15 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10609234
关键词：
Screening identification pericyte specific subpopulation

项目摘要

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.

项目摘要/摘要该应用的长期目的是充分了解周细胞的生物学功能生理和病理状况，并为各种基于周细胞的创新疗法开发与衰老有关的疾病，这与NIA的使命是一致的。该建议旨在解决周围研究领域的关键问题/障碍：缺乏亚群特异性和年龄特定/独立于年龄的周细胞标记。我们建议筛选和识别分子标记使用独特的转基因/生化方法，针对大脑和肌肉周细胞，其次是 RNASEQ分析和随后的创新筛选/比较策略。在AIM 1中，周细胞和血管平滑肌细胞（VSMC）将从大脑和骨骼肌肉中分离出来 AI14：SM22α-CRE小鼠使用我们实验室中优化的基于FACS的方案。由于VSMC- TDTOMATO在此转基因线中的特定表达，这种方法与其他方法不同，允许分离周细胞和VSMC。接下来，新鲜隔离的周细胞和VSMC将受到RNASEQ 分析。首先，针对VSMC的转录剖面将对否定性选择，和富含周细胞的基因将与其他细胞的转录组进行比较（可从公共数据库）。周细胞独有的基因是潜在的周细胞特异性标记。通过比较来自年轻小鼠的周细胞特异性基因，年龄特异性和与年龄无关的标记将是确定。通过比较大脑和肌肉周细胞独有的基因，亚群特异性标记将被确定。在AIM 2中，RNASEQ识别的候选基因将在体外和体内进行验证，并且将评估经过验证的细胞表面标记在基于FACS的周围隔离中的应用。该项目的成功完成不仅将确定特定于周细胞的标记，还将确定亚群特异性（脑部与肌肉）和年龄特异性/与年龄无关（年轻）标记。这些标记将使在体外研究中以高纯度隔离生周细胞，以定位周细胞在体内专门用于功能丧失研究，并在亚型特异性中研究周细胞和特定年龄/年龄与年龄无关的方式。这些研究将极大地丰富我们的知识 Pericyte生物学/功能，生成新的遗传工具，用于新研究线的开门，以及实质上将田地向前推动。