轉化生長因子-β訊號在調控克沙奇病毒和腺病毒受體陽性之肺臟幹細胞肺泡化過程中的雙相作用

Abstract

本研究利用一無血清初代培養 (SFPC) 系統，分離並培養了表達柯薩奇病毒和腺病毒受體 (CAR) 的小鼠肺部幹/祖細胞 (CAR+/mPSCs)，以探討轉化生長因子-β (TGF-β) 訊息在肺泡第一型 (AT1) 細胞分化過程中的時間性作用。研究結果顯示，TGF-β訊息在分化的最初24至48小時內扮演了關鍵且不可或缺的促進角色。在此早期窗口使用阻斷劑A83-01阻斷TGF-β受體I (TGF-βRI) 會顯著影響CAR+/mPSCs向AT1細胞的分化，使細胞停滯於祖細胞或早期過渡狀態。然而，若在分化開始2天後才抑制TGF-β訊息，則對細胞形態分化的影響大為減弱。單細胞RNA定序 (scRNA-seq) 數據進一步證實，與TGF-β訊息及上皮-間質轉化 (EMT) 相關的基因通路，在祖細胞與過渡期細胞中表現活躍，但在分化為成熟AT1細胞後則明顯下調。此先活化後抑制的模式表明，TGF-β訊息的短暫活化對於啟動分化至關重要，而其後續的下調則可能對於防止過度的細胞外基質沉積與纖維化病變至關重要。此外，本研究也觀察到AT1細胞標記物表現的異質性，其中Pdpn表現隨分化時間越久上升，而傳統標記物Ager和Hopx則下降，這可能反映了AT1細胞亞群的存在或體外培養環境的限制。本研究的發現揭示了TGF-β訊息在肺泡上皮分化中精密的時序調控機制，並強調了精準調控TGF-β訊息作用時間，可能成為治療如特發性肺纖維化和支氣管肺發育不全等肺部疾病更具潛力的新策略。

This study utilized a serum-free primary cell culture (SFPC) system to isolate and cultivate mouse pulmonary stem/progenitor cells (CAR+/mPSCs) expressing Coxsackie virus and adenovirus receptors (CAR), with the aim of investigating the temporal role of transforming growth factor-β (TGF-β) signaling in the differentiation process of alveolar type 1 (AT1) cells. The results revealed that TGF-β signaling plays a critical and indispensable promotive role during the initial 24 to 48 hours of differentiation. Inhibiting TGF-β receptor I (TGF-βRI) during this early window significantly impedes the differentiation of CAR+/mPSCs cells into AT1 cells, causing cells to arrest at the progenitor or early transitional stage. However, if TGF-β signaling is inhibited two days after differentiation begins, the impact on cellular morphological differentiation is significantly reduced. Single-cell RNA sequencing (scRNA-seq) data further confirmed that gene pathways associated with TGF-β signaling and epithelial-mesenchymal transition (EMT) were highly active in progenitor and transitional cells but were significantly downregulated after differentiation into mature AT1 cells. This ‘activation followed by inhibition’ pattern suggests that transient activation of TGF-β signaling is crucial for initiating differentiation, while its subsequent downregulation may be essential for preventing excessive extracellular matrix deposition and fibrotic lesions. Additionally, this study observed heterogeneity in the expression of AT1 cell markers, with Pdpn expression increasing with differentiation, while traditional markers Ager and Hopx decreased, potentially reflecting the presence of AT1 cell subpopulations or limitations of the in vitro culture environment. The findings of this study reveal the precise temporal regulation of TGF-β signaling in alveolar epithelial differentiation and emphasize that precise modulation of the timing of TGF-β signaling, may represent a more promising new strategy for treating lung diseases such as idiopathic pulmonary fibrosis (IPF) and bronchopulmonary dysplasia (BPD).