TGF-β誘發小鼠肺部幹/前驅細胞之分化之研究

Abstract

背景： 小鼠肺部幹/前驅細胞 ( mouse pulmonary stem / progenitor cells；mPSC ) 是一種具備新生與分化能力的細胞，可以進行肺部的細胞再生甚至式組織再造，所以肺部前驅細胞的生長與分化是細胞進行增生的關鍵，因此常被認為細胞治療的重要來源。近年來發現隨著肺部發育形成肺泡的過程中，體內TGF-β3會增加，而剔除掉TGF-β3的小鼠出生後擇是活不過20個小時。因此推斷在肺部發育過程中TGF-β3訊息路徑極為可能是肺部前驅細胞驅動分化的重要因子。 目標： 探討TGF-β訊息功能對小鼠肺部前驅細胞之特性維持與生長分化之能力的影響以及其相關機轉之研究。 研究方法： 本篇論文利用體外培養小鼠初代肺部前驅細胞，進一步利用細胞分選儀而得到純化的肺部前驅細胞。接著使用TGF-β拮抗劑, LY364947競爭阻斷TGF-β活化肺部前驅細胞中的Smad-3 路徑。並且藉由分析形成之肺部前驅細胞細胞面積及細胞、細胞數目、細胞形態特、以及細胞特性來評估阻斷TGF-β作用時對於肺部前驅細胞所造成之影響。 結果： 我們從小鼠肺部組織萃取出肺部前驅細胞，並且進一步利用細胞分選儀進行分選得到純化的肺部前驅細胞，並進行體外培養之肺部前驅細胞，研究結果發現，隨著時間的增加原本肺部前驅細胞的標記 ( sox2、nanog、oct-4)會下降，且第一形肺泡細胞 ( T1α、Caveolin-1)的標記會上升，且隨著時間的增加，原本的圓形立方柱狀的細胞會延展攤開且細胞面機驟增。若在培養肺部前驅細胞第一天即給予LY364947阻斷TGF-β / smad3的訊息傳遞，我們發現肺部前驅細胞的面積及細胞特性會維持，且細胞數目相較於未加藥組的明顯得增加；此外，未加藥的控制組則會自發性的分化。 結論： 本篇論文發現，若將TGF-β訊息功能受到LY364947的阻斷時，肺部前驅細胞之特性會增維持住並且抑制肺部前驅細胞趨向分化。這代表TGF-β / smad3對於肺部前驅細胞自發性的分化可能扮演著時分重要的角色，也可能成為未來調控肺部前驅細胞分化或增生特性的重要標的。

Background： mouse pulmonary stem / progenitor cells ( mPSC ) is a kind of cell has the ability for self-renewal and differentiation. Due to mPSC is able to generate new-born cells and repopulate tissue, so the growth and development of mPSC is key of the alveolgenesis. Thus, mPSCs have been considered as an attractive source of cell-based therapy. Recently, it’s been reported that alveolar stage occurred with a burst of TGF-β3 elevation. Therefore, TGF-β3 probably is the main regulator of driving mPSCs into alveolar type 1 cells. Aim： To study the mechanism of TGF-β signaling for the stemness and differentiation potential of mouse pulmonary stem / progenitor cells. Methods： In this thesis, we cultured mPSCs from neonatal lung, and further purified mPSCs through flow cytometer. We used TGF-β receptor inhibitors, LY364947 blocked the activation of smad3 signaling pathway by TGF-β. Then, we analyzed the area and population of mPSCs, cell morphologic of features, and cell expression marker to investigate the effects on mPSCs in the presence / absence of TGF-β signal. Result： We cultured mPSCs through sorting primary culture from neonatal ICR mice lung. The mPSCs underwent spontaneous differentiation. We found that progenitor cells marker ( sox2, nanog, oct-4 ) decreased, and alveolar type 1 cells markers (T1α, Caveolin-1 ) during the spontaneous differentiation. In addition, the cuboidal doom shape colony cells expanded and area per cell increase dramatically. We blocked TGF-β / smad3 signal by LY364947, and found that the cuboidal doom shape, area per cell and the feature of mPSCs maintained. Moreover, the LY364947 group cell number was much more compared to the control group, and the control group underwent spontaneous differentiation. Conclusion： Our results showed that LY364947 once blocking TGF-β / smad3 signaling pathway, the differentiation process was disturbed and the property of the mPSCs maintained. It indicated that TGF-β / smad3 signaling is quite important for mPSCs differentiation, and it could be a great target for regulated mPSCs in the future.