- 學位論文
小鼠肺部上皮前驅細胞誘導血管新生機制之探討
The Study of the Mechanism of Mouse Pulmonary Stem/Progenitor Cells-Derived Angiogenesis
摘要
於先前的研究中,我們發展出一套無血清培養系統,成功地從出生小鼠肺部分離出肺部幹細胞/先驅細胞。這群細胞表現幹細胞之生物標記如: Oct-4, Nanog, Sox-2, 以及 Clara cell分泌蛋白, CCSP。本篇利用初級肺部組織細胞培養之方法篩選出小鼠肺部幹細胞/先驅細胞(mouse pulmonary stem/progenitor cells,簡稱mPSC)。我們分離與純化出的肺部幹細胞/先驅細胞可於有第一型膠原蛋白覆蓋的培養皿中存活數週，並於誘導條件之下可繼而分化成第一型肺泡細胞。先前的研究製備出具有均一大小的明膠生物支架，並將小鼠肺部幹細胞/先驅細胞經10至14天培養後種植於生物支架中進而植入免疫缺陷小鼠的背部皮下組織。實驗結果發現，移植物中觀察到血管新生的現象發生。此外，將種植細胞後的生物支架植入雞胚絨毛尿囊膜( Chick embryo chorioallantoic membrane, CAM)，移植物的組織切片結果顯示肺部幹細胞/先驅細胞可以誘導血管長進生物支架中。上述結果顯示，肺部幹細胞/先驅細胞可能在血管新生的過程中扮演重要角色。於本研究中，我們利用三維細胞培養之技術作為研究肺部幹細胞/先驅細胞之平台，進一步探討細胞於類細胞外基質之環境中是否會參與血管內皮細胞血管新生之過程。首先我們使用管腔形成實驗(tube formation assay)證明肺部幹細胞/先驅細胞主要是透過旁泌作用(paracrine effect)分泌VEGF,FGF等促血管生成因子刺激內皮細胞的管腔形成。另一方面，當我們將肺部幹細胞/先驅細胞與老鼠內皮細胞混和後種植入Matrigel，發現細胞會排列成類似微血管構型的管腔。進一步於共軛焦顯微鏡下觀察發現其內部構造呈中空，與生理環境下之微血管構型極為類似。本研究利用三維之環境進行肺部幹細胞/先驅細胞織培養，提供一個了解肺部幹細胞參與內皮細胞血管新生過程之平台，同時也深入探討肺部幹細胞/先驅細胞在血管新生過程中扮演之角色。
並列摘要
We have previously reported a serum-free primary culture system to generate pulmonary stem/progenitor cells using neonatal mouse lung. The cells expressed stem cell markers, such as Oct-4, Nanog, Sox-4, and Clara cell secretion protein, CCSP. In our research, the pulmonary stem/progenitor cells could be enriched by a primary cells selection culture method. The stem cells we isolated and purified could be kept for weeks on a collagen-I coated plate and underwent terminal differentiation to alveolar type I-like cells in vitro. Meanwhile, we had established a monodisperse gelatin scaffold to co-culture with the pulmonary stem/progenitor cells and transplated subcutaneously into the dorsal body of adult C.B17/Icr-SCID mice. The results suggested that the process of angiogenesis occurred in the construct. In addition, while transplanting the construct into the chick embryo chorioallantoic membrane, the result of HE stained specimens indicated that the pulmonary stem/progenitor cells facilitated the angiogenesis into the construct. Together these results, the pulmonary stem/progenitor cells might play a critical role in the process of angiogenesis. In our research, we use the three-dimensional culture system to serve as a platform for studying the behavior of pulmonary stem/progenitor cells. In extracellular matrix-like environment, further study of whether pulmonary/stem cells would participant in the process of angiogenesis is necessary. In our study, we use the tube formation assay to demonstrate that the pulmonary/stem cells would stimulate the endothelial cell tube forming mainly through secreting proangiogenic factors such as VEGF and FGF. What’s more, while coculture the pulmonary stem/progenitor cell with the murine endothelial cell into Matrigel enable them to form capillary-like tube structure. We further facilitate the confocal microscopy to analysis the structure and find out that the internal construct is similar to the capillary under physiological environment. In our research, we facilitate the three-dimensional culture system as a platform for further studying angiogenesis. Meanwhile, we also intend to investigate whether the pulmonary stem/progenitor cell play a role in the process of angiogenesis.
參考文獻
延伸閱讀
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