第3型乙型轉化生長因子於第Ⅰ型肺泡細胞形態之調節及仿生性氣液相細胞培養於第Ⅰ型肺泡細胞之應用

Abstract

肺泡是呼吸系統中進行氣體交換的場所。肺泡由兩種細胞所構成: 第一型肺泡細胞(type I alveolar cell)為扁平狀細胞，佔肺部總表面積95%以上。上面分布許多微血管，負責肺泡與微血管之間的氣體交換。第二型肺泡細胞(type Ⅱ alveolar cell)成柱狀。可分泌表面張力素(surfactant)，防止肺泡塌陷。 相對於第二型肺泡細胞，第一型肺泡細胞因為缺乏有效的分離方法，使得相關的研究十分有限。而在先前的研究中，我們從新生小鼠的肺部中發現了一群肺臟幹/先驅細胞族群，這群細胞會表現具有特異性的細胞標誌，柯薩奇病毒/腺病毒受體(CXADR)，利用此細胞標誌可以分離出此細胞族群，並將其命名為mPSCsCXADR+。這群細胞在培養七到十天後會分化成近似於第I型肺泡上皮細胞。透過乙型轉化生長因子-3基因剔除小鼠(TGFβ-3 knockout mice)，我們證實了發育過程中TGFβ-3的喪失會抑制第一型肺泡細胞的擴張並進一步導致肺部發育不全。然而，TGFβ-3也將誘導纖維化病理特徵的α-SMA表現量上升，我們的研究發現TGFβ-3在特定時間點的開/關調節對於肺部先驅細胞能否正確分化為第一型肺泡細胞至關重要。 同時，為了能夠更模擬肺部的生理環境，我們將氣液相 (Air-liquid interface, ALI) 細胞培養應用於mPSCs/CXADR+分化的第一型肺泡細胞。我們透過一系列的改良，包括表面張力素並與基質細胞共同培養，從而建立更加適合的仿生ALI模型。我們比較了在不同ALI條件下生長的第一型肺泡細胞，發現在改良後的ALI模型中，第一型肺泡細胞特異性表達的基因表現量以及細胞存活率皆上升。最後，我們預期能將mPSCs/CXADR+分化的第一型肺泡細胞應用致仿生微流體裝置，並重建肺部的生理微環境，以研究第一型肺泡細胞中氣體交換的功能與機制。

Alveoli are tiny balloon shaped structures where gas exchange takes place. It consists of only two types of epithelial cells: alveolar type I II cells. Alveolar type I (AT1) cells are thin and flat in structure, covering 95% of the alveolar surface and involving in the process of gas exchange between alveoli and blood. Alveolar type II (AT2) cells, which are cuboid in shape, release pulmonary surfactant to sustain the surface tension. Although AT1 cells play a critical role in the process of respiration, we have little understanding of the function and regulation of AT1 cells, comparing to its neighbor, the AT2 cells. The limitation of the studies of AT1 cells is due to the lack of a suitable method to isolate the cells for further studies. Recently, we have established a cell model for the studies of AT1 cells. We have identified a rare population of mouse pulmonary stem/progenitor cells (mPSCs), which exclusively expressed a specific cell surface marker coxsackievirus/adenovirus receptor (CAR) in our serum-free culture system. This mPSCsCAR+ were then able to differentiate into AT1-like cells in 7-10 days. In lung development, the process of alveoli differentiation is coupled with a dramatic expansion of the cell surface area. The mPSCsCAR+ derived AT1 cells from transforming growth factor β-3 (TGF-β3) knockout mice indicated that the loss of TGF-β3 during the early stage of lung epithelium differentiation impeded the spreading of AT1cells. Moreover, the expression of TGF-β3 would also induce the expression of α-SMA, which was indicated as a pathologic marker of pulmonary fibrosis. Our studies revealed that TGF-β3 on/off regulation at a critical timing was important for the proper remodeling and expansion of the lung progenitor into AT1 cells. In this study, we also introduced the air-liquid interface (ALI) cell culture to mPSCsCAR+ derived AT1 cells. ALI culture, defined as the basal surface of the cells in contact with liquid culture medium while the apical surface directly exposed to air, is an in vitro organotypic model that can nearly resemble in vivo situations. We made a series of improvement, including the applying of surfactant and co-cultured with stromal cells, to make a more biomimetic model for mPSCsCAR+ derived AT1 cells. We compared the gene expression profile of mPSCsCAR+ derived AT1 cells grown at the different ALI condition, and we found that the gene expression of AT1 cell markers as well as the cell viability was up-regulated. Moreover, we expect to apply the mPSCsCAR+ derived AT1 cells to the biomimetic microfluidic devices, and reconstitute physiological microenvironment in lung to investigate the function and regulation of gas exchange in AT1 cells.