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

• 批准号: 9977608
• 负责人: Yao Yao
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977608
• 关键词: ABCC9 gene; Address; Age; Aging; Alzheimer' s Disease; Biochemical; Biological; Biological Process; Biology; Blood; Blood - brain barrier anatomy; Blood Vessels; Blood capillaries; Brain; Candidate Disease Gene; Cell Separation; Cell surface; Cells; Cerebrum; Databases; Development; Disease; Ectoderm; Genes; Genetic; Genetic Transcription; In Vitro; Innovative Therapy; Investigation; Knowledge; Label; Laboratories; Messenger RNA; Mission; Mus; Muscle; Organ; Pathogenesis; Pathologic; Pericytes; Physiological; Pilot Projects; Population; Population Heterogeneity; Property; Proteins; Protocols documentation; Research; Role; Skeletal Muscle; Smooth Muscle Myocytes; Therapeutic; Transgenic Mice; Transgenic Organisms; Validation; Vascular Smooth Muscle; base; brain tissue; cell type; conditional knockout; design; in vivo; innovation; loss of function; molecular marker; muscle regeneration; mutant; novel marker; screening; single cell sequencing; success; tool; transcriptome; transcriptome sequencing

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.

项目概要/摘要 该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，并开发基于周细胞的创新疗法 与衰老相关的疾病，这与 NIA 的使命是一致的，该提案旨在解决一个问题。 周细胞研究领域的关键问题/障碍：缺乏亚群特异性和年龄相关性 我们建议筛选和鉴定分子标记。 使用独特的转基因/生化方法专门针对大脑和肌肉周细胞，然后 RNAseq 分析和随后的创新筛选/比较策略 In Aim 1, 周细胞和 血管平滑肌细胞（vSMC）将从大脑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 FACS 的协议。 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 接下来，新鲜分离的周细胞和 vSMC 将进行 RNAseq。 周细胞的转录谱将首先针对 vSMC 的转录谱进行负选择， 和周细胞富集的基因将进一步与其他细胞的转录组进行比较（可从 通过比较，周细胞特有的基因是潜在的周细胞特异性标记。 来自年轻和年老小鼠的周细胞特异性基因、年龄特异性和年龄无关的标记将被 通过比较大脑和肌肉周细胞特有的基因，确定了亚群特异性标记。 在目标 2 中，将在体外和体内验证 RNAseq 识别的候选基因，并且 将评估经过验证的细胞表面标记在基于 FACS 的周细胞分离中的应用。 该项目的成功完成不仅将鉴定周细胞特异性标记物，还将鉴定 亚群特异性（大脑与肌肉）和年龄特异性/年龄无关（年轻与年老）周细胞 这些标记物将能够分离出高纯度的活周细胞，用于体外研究，靶向作用。 周细胞专门用于体内功能丧失研究，并研究亚型特异性的周细胞 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，为新的研究领域打开大门，以及 大大推动该领域向前发展。