肺部相关疾病一直是全球高发病率与高死亡率的重要公共卫生难题，其病理特征包括弥漫性肺泡损伤、肺泡–毛细血管屏障破坏、炎性细胞浸润及胶原沉积所导致的结构重塑。现有临床治疗多以对症及支持为主，难以逆转病程，因此，开发能够调节肺部微环境并促进肺组织修复和再生的新型治疗策略已成为研究的重点。
目前干细胞治疗作为再生医学的重要方向，在肺损伤干预中展现出良好前景。然而，直接移植干细胞面临免疫排斥、致瘤风险及归巢效率低等问题。研究表明，干细胞的主要作用机制之一在于旁分泌效应，尤其是经外泌体（Exosomes）介导的信号调控。外泌体是一类直径30–200 nm的细胞外囊泡，富含蛋白质、脂质及核酸，能通过转运功能性分子调节靶细胞信号，从而在组织修复、免疫调节及减缓胶原沉积中发挥作用。相较于细胞移植，外泌体具有低免疫原性、可标准化制备及无致瘤风险等优势，被视为实现干细胞“无细胞治疗（cell-free therapy）”的重要载体。
因此，本研究以人肺干细胞来源外泌体（human lung stem cell-derived exosomes, hLSCs-Exos）为核心，系统探讨其在博来霉素引起的肺损伤修复中的作用与分子机制。研究首先建立了原代人肺干细胞的体外扩增体系，通过qPCR及免疫荧光实验验证其在该培养体系中是否表达干/祖性细胞标志物ID2、NKX2-1、SOX2、SOX9及肺谱系标志物SFTPC，证明其具备干性与肺分化潜能。随后从该细胞培养上清中成功提取并鉴定外泌体，透射电镜（TEM）与纳米颗粒跟踪分析（NTA）显示其粒径为30–200 nm，典型囊泡状结构，并稳定表达外泌体标志蛋白HSP70、CD63、TSG101与CD81，以及肺干细胞特异性标志物SFTPC。
随后我们分别在体外和体内验证该hLSCs-Exos是否具有缓解肺损伤的功能体外实验表明hLSCs-Exos可被人肺上皮细胞（BEAS-2B）有效摄取，显著促进其增殖并减轻博来霉素诱导的细胞损伤。在体内实验中，hLSCs-Exos在小鼠肺组织中可持续富集至少5天，且未造成主要脏器损伤，显示出良好的靶向性与生物安全性。经hLSCs-Exos治疗后，小鼠肺组织的KL-6含量、羟脯氨酸（HYP）含量、促炎细胞因子IL-8与IL-1β以及胶原沉积相关基因Col1a1、Col4a1均显著下降；HE、Masson及天狼星红染色证实hLSCs-Exos能明显减轻肺泡结构破坏和胶原沉积。Western blot结果亦显示纤维化蛋白Col1a1与凋亡蛋白Caspase-3水平显著降低，提示hLSCs-Exos具有有效缓解肺损伤的作用。
为阐明其分子机制，本研究结合外泌体miRNA组学与小鼠组织转录组学进行分析，结果表明hSLCs-Exos中高度富集miR-30d-5p，并确定其靶基因为血小板反应蛋白Thbs2。进一步实验显示，miR-30d-5p过表达可下调Thbs2及其下游p-Akt水平，抑制Caspase-3与Col1a1的表达，说明其通过miR-30d-5p/Thbs2/Akt信号轴调控炎症、凋亡与纤维化过程。由此可见，hLSCs-Exos通过转运miR-30d-5p抑制Thbs2，进而调控Akt信号通路，从而实现对肺损伤的保护与修复。
综上，本研究系统建立了人肺干细胞与其外泌体的培养与鉴定平台，全面验证了hLSCs-Exos在急性肺损伤修复中的安全性与疗效。研究首次揭示了miR-30d-5p–Thbs2–Akt信号轴在外泌体介导肺修复中的关键作用，为肺损伤等疾病的无细胞治疗提供了新的理论依据与分子靶点。

Lung-related diseases remain a significant public health challenge with high morbidity and mortality rates globally. Lung injury is characterized by diffuse alveolar damage, disruption of the alveolar-capillary barrier, inflammatory cell infiltration, and structural remodeling due to collagen deposition. Current clinical treatments are primarily symptomatic and supportive, and often fail to reverse disease progression. Therefore, developing novel treatment strategies that can modulate the lung microenvironment and promote lung tissue repair and regeneration has become a key research focus.
Currently, stem cell therapy, as an important direction in regenerative medicine, shows promising promise in lung injury intervention. However, direct stem cell transplantation faces challenges such as immune rejection, tumorigenesis risk, and low homing efficiency. Research indicates that one of the main mechanisms of stem cell action is paracrine effect, particularly exosome-mediated signal regulation. Exosomes are extracellular vesicles with a diameter of 30–200 nm, rich in proteins, lipids, and nucleic acids. They can regulate target cell signaling by transporting functional molecules, thereby playing a role in tissue repair, immune regulation, and slowing collagen deposition. Compared to cell transplantation, exosomes possess advantages such as low immunogenicity, standardized preparation, and no tumorigenic risk, making them a crucial carrier for achieving cell-free stem cell therapy.
Therefore, this study focuses on human lung stem cell-derived exosomes (hLSCs-Exos) to systematically explore their role and molecular mechanisms in the repair of bleomycin-induced lung injury. The study first established an in vitro expansion system for primary human lung stem cells. qPCR and immunofluorescence experiments were used to verify whether these cells expressed stem/progenitor cell markers ID2, NKX2-1, SOX2, and SOX9, as well as the lung lineage marker SFTPC, within this culture system, demonstrating their stem cell and lung differentiation potential. Exosomes were subsequently successfully extracted and identified from the cell culture supernatant. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that the particle size was 30–200 nm, with a typical vesicle-like structure, and stably expressed exosome marker proteins HSP70, CD63, TSG101, and CD81, and specific marker of hLSCs SFTPC.
We then validated the function of hLSCs-Exos in alleviating lung injury in vitro and in vivo. In vitro experiments showed that hLSCs-Exos could be effectively taken up by human lung epithelial cells (BEAS-2B), significantly promoting their proliferation and alleviating bleomycin-induced cell damage. In in vivo experiments, hLSCs-Exos could be continuously enriched in mouse lung tissue for at least 5 days without causing damage to major organs, demonstrating good targeting and biocompatibility. Following treatment with hLSCs-Exos, mouse lung tissue showed significant decreases in KL-6 levels, hydroxyproline (HYP) levels, pro-inflammatory cytokines IL-8 and IL-1β, and collagen deposition-related genes Col1a1 and Col4a1. HE, Masson's red, and Sirius red staining confirmed that hLSCs-Exos significantly reduced alveolar structural damage and collagen deposition. Western blot results also showed significantly reduced levels of the fibrotic protein Col1a1 and the apoptotic protein Caspase-3, suggesting that hLSCs-Exos effectively alleviates lung injury.
To elucidate its molecular mechanism, this study combined exosomal miRNA analysis with mouse tissue transcriptomics. The results showed that hLSCs-Exos was highly enriched in miR-30d-5p, and its target gene was identified as the platelet-reactive protein Thbs2. Further experiments showed that overexpression of miR-30d-5p downregulated Thbs2 and its downstream p-Akt levels, and inhibited the expression of Caspase-3 and Col1a1, indicating that it regulates inflammation, apoptosis, and fibrosis through the miR-30d-5p/Thbs2/Akt signaling axis. Therefore, hLSCs-Exos inhibits Thbs2 by transporting miR-30d-5p, thereby regulating the Akt signaling pathway and achieving protection and repair of lung injury.
In summary, this study systematically established a platform for the culture and identification of human lung stem cells and their exosomes, comprehensively validating the safety and efficacy of hLSCs-Exos in the repair of acute lung injury. This study is the first to reveal the crucial role of the miR-30d-5p–Thbs2–Akt signaling axis in exosome-mediated lung repair, providing new theoretical basis and molecular targets for cell-free therapy of lung injury and other diseases.