肺间质特异性缺失Fstl1导致小鼠肺泡形成异常的分子机制

Alveologenesis, the final step of lung development, is characterized by the formation of ～95% adult gas-exchange surface area and is essential for normal breathing. The genetic network driving alveologenesis is poorly understood compared with earlier steps in lung development. Fstl1 is a lung secreted protein modulating lung development, tissue homeostasis and lung diseases. Our previous studies observed that mesenchymal Fstl1 is important for alveologenesis. While conventional KO mice exhibit neonatal lethality, Fstl1 mesenchymal cKO mice (E15.5-18.5) are born phenotypically normal. However, they exhibit defects in alveologenesis characterized by an emphysema-like phenotype in early postnatal stage that becomes more pronounced at adult stage. We hypothesize that Fstl1 regulates alveolar myofibroblast behavior in a spatiotemporal manner, mediating the formation of elastin-aSMA network during alveologenesis. In this project, we will use cKO mice, lineage-deletion mice, setup elastase-induced emphysema mouse model, in combination of genetic lineage tracing, single-cell RNA sequencing, and primary cell culture approaches, to study the mechanisms underlying the myofibroblast-driven septal crest formation, and to score the recombinant Fstl1 protein for potential intervention of disease process. Our study will improve the understanding of the molecular mechanisms underlying alveogenesis, and may offer potential therapeutic targets for patients with emphysema.

出生后肺泡形成是肺发育的最后阶段，负责成人约95%换气面积的生长，对正常呼吸至关重要。相对于早期肺发育，调控肺泡形成的遗传网络知之甚少。糖蛋白Fstl1在肺发育、损伤修复和疾病中发挥重要作用。课题组前期发现间充质来源的Fstl1参与小鼠肺泡形成。对比全敲小鼠出生致死，肺间质（E15.5-18.5）Fstl1敲除小鼠出生正常，但出生后肺泡异常增大，类似肺气肿表型，成年时愈加严重。本课题将利用条件性基因敲除小鼠和谱系删除小鼠，建立弹性蛋白酶诱导的肺气肿小鼠模型，结合谱系追踪、单细胞测序和原代细胞培养等技术，对肺泡形成的关键环节：肌成纤维细胞驱动隔嵴形成的机制深入探讨，以期取得进展并转化为临床应用。目的揭示Fstl1时空特异性调控肌成纤维细胞的行为和功能，参与elastin-aSMA纤维网络建成，在肺泡形成中起重要作用。本研究将提升对肺泡形成机制的认识，为肺气肿的临床治疗提供潜在靶点。

肺泡肌成纤维细胞（aMYF）参与肺泡二次分隔，是出生后的肺泡期中肺泡形成的重要效应细胞。但其特异细胞标志物未知，细胞来源和分隔调控功能尚未清楚，期待新的研究突破。本项目通过分析肺间充质特异性敲除Fstl1基因的小鼠（Fstl1MCKO）肺泡简化的表型，结合scRNA-seq分析技术，发现myoFB的一个亚群（MyoFB_1），Fstl1缺失导致的细胞群数量减少与肺泡简化相关，我们将其定义为aMYF，并确定细胞标志物（Pdgfra+Tgfbi+Trim67+Cdh4++），细胞来源为气道平滑肌细胞（ASM）。进一步实验证实Fstl1调控的TGF-b1信号通过调控aMYF的elastin组装功能，参与elastin网络模式建立和肺泡形成。Fstl1缺失可导致肺泡简化，表明Fstl1MCKO 小鼠是新生儿肺部疾病BPD新的小鼠遗传模型， Fstl1是BPD临床治疗的潜在靶点。我们的研究拓展了人们对肺泡形成过程和机制的认识，为深入研究BPD的病理机制提供理论基础。