小鼠肺部幹細胞與具有均一孔洞之明膠生物支架於肺部組織工程上之應用

Abstract

在本研究中，我們製備有均一孔徑之明膠生物支架將小鼠肺部幹細胞/先驅細胞（pulmonary stem/progenitor cell）種植於其中並進行細胞培養，於體外培養以及植入免疫缺陷小鼠（SCID mice）體內皆能形成肺泡狀的結構，於先前的研究中，我們利用無血清細胞培養成功地從初生小鼠肺部分離出肺部幹細胞/先驅細胞。此細胞表現出幹細胞之生物標記如：Oct-4、Nanog、Sox-2、以及CCSP（Clara細胞分泌蛋白），顯示此細胞為Clara細胞之亞群，而Clara細胞於肺部損傷的動物模型中一直以來被認為是肺部幹細胞/先驅細胞。我們分離與純化的初代培養細胞能夠在體外培養的環境中存活數週，同時也具有細胞分化的能力，於體外培養的環境下能夠進一步分化成肺泡第二型細胞（alveolar type II cell）與肺泡第一型細胞（alveolar type I cell）。此結果顯示，於組織工程學上，此肺部幹細胞/先驅細胞具有在體外進行細胞培養進而形成類肺泡組織的潛力。於本研究中，我們利用微流體通道所製備具有均一大小的明膠微氣泡（gelatin microbubble），在一定的氣體壓力以及適當的明膠液體流速下，能夠形成穩定的明膠微氣泡，收集堆疊整齊的微氣泡後成膠、交連、抽真空後所得之生物支架（scaffold）進行三維的細胞培養。透過控制微流體通道之大小氣體壓力以及液體流速，能夠製備出具有不同直徑之微氣泡，本研究中成功的製備出50微米（μm） 至200 微米等不同孔徑之生物支架。此具有高孔隙以及海綿狀的生物支架結構非 常類似肺泡組織。本研究欲應用此肺部幹細胞/先驅細胞與均一孔徑之生物支架 以發展肺部組織工程於再生醫學。 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後，再經兩 週的培養，以共軛焦顯微鏡、組織切片、以及電子顯微鏡能夠發現肺泡幹細胞/ 先驅細胞於生物支架中形成肺泡狀的結構，同時利用反轉錄聚合酵素鏈鎖反應以 及細胞免疫螢光染色發現細胞表現出肺泡第二型細胞與肺泡第一型細胞。此結果 顯示肺部幹細胞/先驅細胞種植於生物支架後，不僅能夠形成肺泡狀的結構，也能被誘導分化成肺泡細胞。於動物模式的實驗中，我們將種植細胞的生物支架植 入免疫缺陷小鼠的背部皮下組織，於生物體內觀察肺部幹細胞/先驅細胞的表現。 植入後一週、二週、四週後取出，也同樣的呈現肺泡狀的結構，表現出肺泡細胞 的特定蛋白。利用綠色螢光小鼠培養出的肺部幹細胞/先驅細胞，有利我們觀察 細胞於受植入的小鼠體內存活的情況，實驗發現，植入的細胞至少能夠在免疫缺 陷小鼠皮下組織存活四週。移植物裡也觀察到血管新生的現象發生，犧牲小鼠前 透過尾靜脈注射dextran-FITC 進行血管染色，於螢光顯微鏡下，能觀察移植物 中的血管新生，比較植入不含細胞的生物支架或含細胞之生物支架，結果顯示含 細胞者生成較多的血管，肺部幹細胞/先驅細胞可能在血管新生中也扮演著重要 的角色。 本研究結果顯示利用具有均一孔徑的明膠生物支架進行三維的肺泡幹細胞/ 先驅細胞培養，提供了肺臟細胞一個適當的微環境生長。經此組織工程培養後的 人工肺泡組織不僅具有肺泡狀的結構，同時也分化出特定的肺泡細胞，在未來具 有發展肺部組織工程，以及臨床上利用組織工程進行再生醫學治療的潛力。

In our research, we demonstrated that the pulmonary stem/progenitor cells within monodisperse gelatin-microbubble scaffold could create alveoli-like construct in vitro and in vivo. In previous study, we have reported a serum-free primary culture system to generate pulmonary stem/progenitor cells from neonatal mouse. The cells expressed stem cell markers, such as Oct-4, Nanog, Sox-2 and CC10, and also Clara cell secretion protein indicating they represented a sub-population of Clara cells which have long been implicated as pulmonary stem/progenitor cells in lung injury models. These stem cells could be kept for weeks in the primary cultures and underwent terminal differentiation to alveolar type II (AT2) and type I (AT1) like cells sequentially in in vitro differentiation. The results displayed that the stem/progenitor cells hold the potential promise to grow pulmonary tissue in vitro. In the study, the microfluidic method was applied to production of monodisperse gelatin-microbubble scaffold. Under the stable formation of bubble regiem, monodisperse gelatin-microbubbles modulated by varying the flow rate, gas pressure, and channel size. The microbubble scaffolds, the size ranging from 50μm to 200μm, were seeded with pulmonary stem/progenitor cells to explore the application potential for stem cell-based tissue engineering. After 10 to 14 days’ culture, the scaffold showed porous and spongy structures similar to alveolar units. The structure for the scaffold and the cells were examined by RT-PCR, immunohistochemistry and electromicroscopy. Cells forming “alveolar-like structures” in the inner sites of the microbubbles were stained positive for alveolar AT2 and AT1 cells by immunohistochemical analysis and RT-PCR, and there were still a few stem/progenitor cells keeping their phenotype in the structure. The results indicated that the pulmonary stem/progenitor cells could be induced to the terminal differentiation for AT2-like and AT1-like cells as well as the promotion of three-dimensional alveolar tissue growth via the gelatin-microbubble scaffold. In animal model, we utilized the subcutaneous transplantation model on in vivo generation of vascularized pulmonary tissue constructs. The constructs were transplated subcutaneously into the dorsal body of adult C.B17/Icr-scid mice to facilitate in vivo pulmonary tissue construct formation. After transplantation, the constructs showed “alveolar-like structures” in scaffold with positive immunohistochemical staining for epithelial and endothelial cells. The pulmonary cells of GFP transgenic mice in the constructs were able to survive in the subcutaneous tissue of adult mice for at least 28 days. Angiogenesis of the constructs was demonstrated with tail vein injection of fluorescein isothiocyanate–conjugated dextran. In the scaffold-only controls, some level of host infiltrate, but no measurable vascularization, was detected. This study suggests that the three dimensional cell culture system provided an appropriate microenvironment for pulmonary cells, and demonstrates that the cell-based tissue engineering possessed the potential promise to regenerate alveolar-like structures for the development of engineering lung tissue for clinical applications.