New Approaches for the Study of Lung Fibrocytes

纤维细胞被认为是一种独特的骨髓来源细胞，有助于胶原蛋白的积累。 在肺损伤期间。纤维细胞被定义为CD45+/I型Col I+，强调其推定的， 同时具有造血型和结缔组织表型。用于以下目的的方法的局限性 然而，鉴定和分离纤维细胞以及对其基本生物学的了解不足， 限制了该领域的进展，并加剧了人们对它们在肺纤维化反应中所起作用的怀疑。至 由于以下事实，用于研究纤维细胞的方法主要提供了间接信息 纤维细胞鉴定和分离技术不能直接产生活细胞；在这方面 分离需要在体外长期培养单核细胞。作为回应，我们开发了一种 直接分离可存活的纤维细胞的战略，并正在寻求R21资金，以使用这项技术 推进这一领域，为今后的工作奠定基础。为此，我们使用了一种转基因 在I型Col启动子(Col-GFP)控制下表达GFP的小鼠。从这只鼠标， CD45+/GFP+细胞可用流式细胞仪分离。值得注意的是，GFP+细胞活跃在 转录Col mRNA；因此，CD45+/GFP+细胞表现出经典定义的纤维细胞的特性。 在正常肺和博莱霉素处理的5d肺中，纤维细胞占肺总CD45+细胞的8-9%。 并表达髓系标志物。随着损伤的发生，肺的绝对数量增加了~2倍 纤维细胞伴随着I型Col基因mRNA的15倍增加。这些发现提高了 基本问题：损伤中纤维细胞的扩张和激活是局部的还是招募的？ 细胞？为了回答这个问题，我们还开发了一种胸部屏蔽方法，可以保护 移植期间驻留的肺CD45+细胞，因此支持嵌合体的发育 肺CD45+和骨髓CD45+细胞群各自表达不同的荧光 记号笔。拥有屏蔽模型和Col-GFP小鼠提供了一个独特的机会 回答这一基本问题，同时建立肺成纤维细胞知识库 其有效性不受技术问题的影响。因此，我们建议提供基本的 关于它们的定位和个体发育的信息(目标1)，以检验它们是 稳定的常住人口(目标1)，以检验胶原蛋白激活在常住居民中发生的假设 肺损伤过程中的纤维细胞(目标2)，并产生2个比较基因组签名，将 提供有关肺纤维细胞特性、新的候选标记物和调节的关键信息 控制其激活的途径(目标2)。我们的长期目标是为 确定肺纤维化中纤维细胞命运和功能的试剂的基本原理设计。

Fibrocytes are thought to be a distinct marrow-derived cell that contributes to collagen accumulation during lung injury. Fibrocytes are defined as CD45+/type I Col I+, underscoring their putative, simultaneous hematopoietic and connective tissue phenotype. Limitations in methods used to identify and isolate fibrocytes along with deficiencies in understanding their basic biology, however, restrict progress in the field, and fuel skepticism regarding their role in lung fibrogenic reactions. To date, methods used to study fibrocytes have primarily provided indirect information due to the fact that fibrocyte identification and isolation techniques do not yield viable cells directly; in this regard isolation involves prolonged culturing of monocytic cells in vitro. In response, we developed a strategy to directly isolate viable fibrocytes, and are seeking R21 funding to use this technology to advance the field and establish a foundation for future work. For this, we employ a transgenic mouse that expresses GFP under control of the type I Col promoter (Col-GFP). From this mouse, CD45+/GFP+ cells can be readily isolated by flow cytometry. Of note, GFP+ cells are actively transcribing Col mRNA; thus CD45+/GFP+ cells display properties that classically define fibrocytes. In the normal and 5 d bleomycin-treated lung, fibrocytes comprise ~8-9% of total lung CD45+ cells and express myeloid markers. With injury there is a ~2-fold increase in the absolute number of lung fibrocytes accompanied by a 15-fold increase in type I Col gene mRNA. These findings raise the basic question: is the expansion and activation of fibrocytes in injury localized to resident or recruited cells? To answer this question, we also developed a thoracic shielding method that protects resident lung CD45+ cells during transplantation, and as a result, supports development of chimeras with lung CD45+ and marrow CD45+ cell populations that each expresses a different fluorescent marker. Having the shielding model and Col-GFP mice in hand provides a unique opportunity to answer this basic question, and at the same time to develop a knowledge base for lung fibrocytes whose validity is not undermined by technical concerns. We, thus, propose to provide basic information regarding their localization and ontogeny (Aim 1), to test the hypothesis that they are a stable resident population (Aim 1), to test the hypothesis that collagen activation occurs in resident fibrocytes during lung injury (Aim 2), and to generate 2 comparative genomic signatures that will provide key information about lung fibrocyte identity, new candidate markers, and regulatory pathways controlling their activation (Aim 2). The long-term objective is to lay the groundwork for the rationale design of reagents to definitively resolve fibrocyte fate and function in lung fibrosis.