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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.

项目总结/摘要本申请的长期目标是充分了解周细胞在肿瘤中的生物学功能。生理和病理条件，并开发基于周细胞的各种创新疗法衰老相关疾病，这与NIA的使命一致。该提案旨在解决周细胞研究领域的关键问题/障碍：缺乏亚群特异性和年龄- 特异性/与年龄无关的周细胞标记物。我们建议筛选和鉴定分子标记使用独特的转基因/生物化学方法特异性针对脑和肌肉周细胞，然后 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）将从以下动物的脑和骨骼肌中分离： Ai 14：SM 22 α-Cre小鼠使用我们实验室优化的基于FACS的方案。由于vSMC- tdTomato在该转基因品系中的特异性表达，与其他方法不同，该方法允许分离周细胞和血管平滑肌细胞接下来，将对新鲜分离的周细胞和vSMC进行RNAseq 分析。周细胞的转录谱将首先针对vSMC的转录谱进行负选择，并且将周细胞富集的基因与其它细胞的转录组（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。通过比较来自年轻和老年小鼠的周细胞特异性基因，年龄特异性和年龄无关性标记物将被鉴定通过比较大脑和肌肉周细胞特有的基因，将被识别。在目标2中，将在体外和体内验证RNAseq鉴定的候选基因，将评估经验证的细胞表面标记物在基于FACS的周细胞分离中的应用。本项目的成功完成不仅将确定周细胞特异性标记，还将确定亚群特异性（脑与肌肉）和年龄特异性/年龄无关性（年轻与老年）周细胞标记。这些标记物将能够以高纯度分离活周细胞用于体外研究，靶向周细胞专门在体内的功能丧失的研究，并研究周细胞在一个亚型特异性和年龄特异性/年龄独立性的方式。这些研究将极大地丰富我们的知识，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，大大推动了这一领域的发展。