調控唾腺球之結構和來源以探討其促進唾液腺再生之潛力

Abstract

唾腺功能低下是一長期未解的問題，在臨床上以“口乾症 (xerostomia)'的形式表現。唾腺分泌唾液功能的缺失會使得生活品質受到影響。截至目前，細胞治療或許是一種有效治療唾腺功能低下的治療策略。因此，開發細胞來源對於細胞治療的可行性是一重要的議題。唾腺球(Salispheres)是由唾腺幹細胞所衍生之球形細胞微結構。在本研究中，我們首先從不同解剖位置之三對主要唾腺中分離並培養唾腺球，其中包括下頷腺 (submandibular glands)、耳下腺 (parotid glands)與舌下腺 (sublingual glands)。我們接著解析不同來源唾腺球中的細胞特性、幹細胞特性、以及體外培養之分化能力。研究結果指出不同來源的唾腺球表現出不同的細胞特性但仍具有高度相似地幹細胞特性。接著，我們觀察到唾腺球的體外自發性分化(In-vitro spontaneous differentiation)是以細胞球體空腔化的形式呈現並伴隨著幹細胞特性的流失。接著，我們將生醫材料應用於唾腺球的培養系統中並進一步探索唾腺球在不同生醫材料微環境中的細胞行為。我們指出以幾丁聚醣(chitosan)為基底的生醫材料具有保留唾腺球幹性的能力，此一能力是幾丁聚醣透過調控唾腺球之細胞骨架重組並影響細胞極化而非細胞凋亡(apoptosis)與程序性死亡所致。試驗結果證實幾丁聚醣的培養系統能夠有效維持唾腺球之原始結構與幹性並同時證實以幾丁聚醣為基底的生醫材料在組織工程的應用潛力。我們接著藉由腺體內細胞球移植來解析唾腺球在體內之治療效果，其結果指出不同來源之唾腺球皆能有效改善放射線照射過之唾腺並修復分泌唾液的能力。在不同來源之唾腺球移植的唾腺中，我們觀察到典型唾腺組織結構的恢復與纖維化微環境的重塑現象，這些結果暗示不同對唾腺之功能贅餘的特性，並有助於未來如何設計細胞治療用於改善功能性低下之腺體器官。

Dysfunctional salivary gland (SG) is a long-term unresolved problem and is presented as xerostomia in clinical. Lack of saliva producing capability of SG hampered the improvement of quality of life. To date, cell therapy is a promising therapeutic strategy for gradually restoring SG function. Hence, exploration of cellular sources is critical. Salispheres are spheroid cellular organizations that derived from SG stem cells. We first generated salispheres from three major pairs of SGs which located in different anatomic regions including submandibular glands (SMG), sublingual glands (SLG) and parotid gland (PG). In the current study, we investigated the cellular features, stemness properties, and in-vitro differentiation capability within major SGs-derived salispheres. Our results demonstrate that salispheres exhibit distinct cellular features but share similar stemness properties. Subsequently, in-vitro spontaneous differentiation of salispheres are observed and presented as spheroid cavitation which further lead to loss of stemness during in-vitro expansion. Therefore, applications of biomaterials are involved to further explore the cellular behavior upon distinct biomaterials. Chitosan-based biomaterials are then demonstrated to be capable of maintaining stemness properties of salispheres by regulating the cytoskeleton re-arrangement and polarity establishment but not apoptosis and programmed cell death. The results suggest that the chitosan-containing system could effectively maintain the primitive structures and properties of salispheres which demonstrates the potential application of chitosan-based biomaterials for further tissue-engineering demands. The in-vivo effects of major SGs-derived salispheres are investigated by intra-glandular transplantation. The results revealed that salispheres are all capable of improving the saliva secreting ability of irradiated SMG. Typical histological structures can be observed in transplanted glands and the fibrotic microenvironments are remodeled by transplanting salispheres regardless of origins. The results hint the possibility of functional redundancy upon distinct pairs of major SGs, which is helpful for the design of cell therapy to rescue the dysfunctional glandular organs.