人類唾腺細胞：分離、擴增與功能

Abstract

唾腺 (salivary gland) 位於消化道的起點，並負責唾液(saliva)的分泌。唾液負責維持口腔內的濕度以及含有許多種類的電解質與蛋白質用以進行初步的消化功能。因頭頸部腫瘤而給予的放射線治療與自體免疫疾病會引起唾腺組織失去功能，造成乾口症以及許多併發症。解決此問題的有效手段應是經由組織工程建立一個具有自分泌功能的裝置。要達到此目標，最大的困難點為獲取適當的初代培養 (Primary culture) 細胞。其原因為唾腺中之細胞皆位處高度分化的階段，在體外環境要進行大量增殖的培養與誘導分化刺激有很高的困難度，也導致了這些腺體細胞在體外環境階段的相關文獻相當缺乏 。因此一個用以分離與增加唾腺中不同種類的細胞族群的穩定培養程序是非常需要的。本研究之目標在於建立一個用於獲取唾腺細胞的穩定培養系統，而後對其功能的表現做鑑定與觀察，期望能做為進行組織工程之用途。 本研究分為三大部份，第一部份，我們以培養基選擇法找到了適合於不同種類的唾腺細胞的培養條件，並且自基因到蛋白質層次鑑定了這些細胞的特性。這些具有不同型態的細胞被從人類的唾腺組織中分離出來，並且擴增細胞數量。這系統只經由簡單且固定的組織處理手續便可達成從同一批檢體中同時以高純度培養出三種不同種類的細胞，分別為：腺泡(Acinar, PGAC)細胞，肌上皮(Myoepithelial, PGME)細胞，以及纖維母細胞 (Fibroblast, FB)。 我們鑑定了這些初代培養細胞的特性，以準備將來的實驗平台應用。 第二部份討論我們培養的唾腺細胞在培養過程中出現的聚集行為。本研究利用無血清的培養基在低鈣離子濃度的條件下培養出腺泡細胞，此細胞能持續增殖放大數量，並在此同時保持著腺泡細胞的特性，例如分泌唾液澱粉酶 (α-amylase)，表現水通道蛋白 (Aquoporins) 與緊密連接(Tight junction)結構相關的蛋白分子 (ZO-1)等。值得注意的是，這些細胞在滿盤或是處在高細胞密度的狀況下時，會形成三維(Three-dimension)的立體結構。我們稱這些結構為滿盤後結構 (Post-confluence structure)。雖然培養的環境與培養基並未改變，但是這些立體結構中的細胞較二維(Two-dimensional)表面上的細胞有較高的功能性蛋白質分子表現。另外，分子位置轉移的現象也出現在水通道蛋白與緊密連結蛋白。這些結果告訴我們，雖然沒有使用複雜的三維支架(Scaffolds)或是其他細胞外間質蛋白(Extra-cellular matrix protein)來刺激這些細胞，他們仍然具有啟動分化動作的表現。 第三部份延伸第二部份所觀察到的滿盤後結構之研究，我們引進了聚乳酸-甘醇酸 (poly(lactic-co-glycolic acid), PLGA)做為培養基材，來與先前實驗中所用的組織培養用聚苯乙烯(Tissue culture polystyrene, TCPS)來做比較，觀察是否能藉由不同性質的基材來控制腺泡細胞形成滿盤後結構的行為。結果顯示在聚乳酸-甘醇酸上的滿盤後結構的大小與數量與在組織培養用聚苯乙烯上相比皆有明顯較發達的現象。本研究發現滿盤後結構的形成原因與於鈣黏著素E (E-cadherin)的功能表現有關，而兩種材料上滿盤後結構不同的形成傾向則是由於鈣黏著素E在兩種基材上有著不同程度的調節所導致的。在此實驗系統中，我們也發現這個調節是經由FAK-ILK-Snail這條訊息傳導路徑產生的影響。 經由我們的研究結果，我們提出了一個穩定的操作條件來對人類唾腺細胞來進行初代培養，並證實細胞產生具分化現象結構的傾向可藉由使用不同特性的基材來達成。

Salivary glands locate at the start of digestive system and are responsible for the secretion of saliva, which has many functions in maintaining homeostasis of the oral cavity. However, radiotherapy and autoimmune diseases may cause dysfunction of these glands and lead in the desiccant oral environment and many side effects. An engineered auto-secretary device will be the ultimate solution for this condition. As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of not only reports on the in vitro cell biology and physiology of human salivary gland cells but also proper cell source. Complexity of the gland explains the urgent demand for a reliable protocol to isolate and expand various gland cells that can be used for further study. This study aims to set up a stable protocol for primary cultivation of human salivary gland cells from surgery specimens and further introduce those cells into tissue engineering. In the first part, respective optimal culture conditions for three types of gland cells were set up by medium selection and then identified the phenotypes from mRNA to protein level. Those cultured cells with different morphologies were isolated and expanded without complex mechanical processes and expensive techniques such as flow cytometry sorting. The harvested cells were acinar (PGAC) cells, myoepithelial (PGME) cells, and fibroblasts (FBs). The proposed protocol is simple with a high success rate to culture various gland cells, making it highly promising for use in future tissue engineering studies. The second part discusses the aggregating behavior of PGAC cells. This study used the serum-free and low-calcium culture system to obtain PGAC cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporins-5, and ZO-1). Notably, when the cells reached confluence, 3-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the 2-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form aggregations and exhibit certain level of differentiating characteristics without the help of 3D scaffolds. This phenomenon implies that introducing 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering is of potential. Extending the phenomenon of PCS formation that was discussed in the second part, the third part aims to investigate if the culture substrates have the effects on controlling cells in this aggregating process. Under serum-free environment, primary human PGAC cells can be obtained. After reaching confluence, PGAC cells spontaneously form 3D cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffolds.