人類唾液腺組織工程之研究：建立幹細胞與共培養系統

Abstract

唾液腺是人體外分泌腺的一種，主要在掌管唾液的分泌，在分泌的唾液中，除了水分外還含有電解質、黏液、分泌蛋白、酵素與生長因子。唾液的功能在幫助吞嚥和消化食物，也可以維護口腔健康，因此唾液腺功能低下的患者其生活品質會受到很大的影響，唾液腺功能損失的原因很多，可能由自體免疫引起的唾液腺發炎和組織破壞，也可能因為接受醫療處理而導致，例如頭頸部腫瘤病患在進行放射線治療後，唾液腺功能會受到影響，這些病人因無充足的唾液分泌，常常會有口乾的症狀，伴隨吞嚥和消化困難、齲齒、吸收不良、口腔黏膜受損等症狀，對生活產生許多不便和健康的危害。然而，目前在醫學上並無有效的方法來治療放療引起的唾液腺損傷，藉由組織工程的方法重建新的唾液腺，是這些病人未來的希望。 組織工程的發展上，細胞層面是最基礎需解決的問題，因此在本研究中，分析唾液腺分泌細胞從腮腺組織分離培養過程中的變化，在培養至第14天可以取得高比例的唾液腺幹細胞。目前研究上，對幹細胞的篩選多用流式細胞儀針對特定表面抗體標記，或是利用創傷刺激幹細胞比例增加，在本研究中分析唾液腺細胞培養過程中，培養一至兩週的細胞，利用CD49f與CD90標記進行分析，幹細胞比例顯著上升，且進一步證實細胞具有乙醛脫氫酶1 (aldehyde dehydrogenase isoform 1, ALDH1)表現與分化能力。長期培養三至四週後，可以獲得穩定的唾液線分泌上皮細胞，並具有分泌細胞的水通道蛋白表現，但在培養四週的時間點下，培養的細胞無法經由誘導產生脂分化與骨分化，ALDH1、幹細胞表現的表面標的表現也隨之下降。蛋白質表現顯示剛開始貼附生長的細胞會喪失上皮細胞的特性，而隨著培養天期增加才逐漸恢復E-chadherin表現，推測此處理過程細胞經歷間質-上皮轉化(mesenchymal-epithelial transition, MET)，在這項實驗中，證實了我們的培養系統中唾液腺幹細胞的存在，並且建立一個簡單的方法取得充足的唾液腺幹細胞以利組織工程應用。 隨著唾液腺分泌細胞在培養系統中的生長，功能的維持是下一階段的目標。已知間質細胞在組織的形成與發育過程中扮演著非常重要的角色，因此，要成功地利用組織工程技術建構人工唾液腺，間質細胞與分泌細胞間複雜的互動關係有其重要性。我們將分離出的分泌細胞與纖維母細胞進行共培養，並探討細胞彼此間之互動關係。研究中發現，分泌細胞與纖維母細胞進行直接的共培養於同一培養環境，分泌細胞會聚集為一個個細胞群落，而纖維母細胞圍繞在週邊，在這樣的生長情境下，分泌細胞所製造的唾液澱粉酶顯著上升，此上升幅度與纖維母細胞數目和分泌的basic fibroblast growth factor (bFGF)有關，分泌細胞受到此生長因子影響，刺激唾液澱粉酶製造。使用纖維母細胞的條件培養液也有相似的效果，更確立唾液澱粉酶製造受到纖維母細胞分泌的生長因子影響。除此之外，我們發現不同生醫材料對纖維母細胞的生長因子分泌亦有不同的影響，因而對影響分泌細胞製造唾液澱粉酶有不同程度的改變，其中針對一些不同特性的材料做研究，發現在這些材料中，纖維母細胞被聚偏氟乙烯(polyvinylidene fluoride, PVDF)調控後的條件培養液對分泌細胞產生的澱粉酶有最顯著的提升。由唾液腺分離出來的纖維母細胞在PVDF影響下，會產生部分神經營養因子neurotrophin-4 (NT-4)，先前研究已知，神經發展在唾液腺發育中佔有重要的地位，在此PVDF調控的纖維母細胞條件培養液培養的分泌細胞，不但細胞內製造的唾液澱粉酶顯著上升，其分泌至細胞外的唾液澱粉酶活性亦有明顯增加。 細胞在體內環境中受到許多因子的調控，這是在離體環境中很難重現的，針對唾液腺分泌細胞與周圍的間質細胞研究，初步在成熟的分泌細胞培養環境中加入由周邊細胞主動分泌的生長因子調控，可對唾液腺分泌細胞、間質細胞以及生醫材料間互動關係有了基礎的瞭解。然而利用組織工程重建唾液線組織的功能是一個重要且相當複雜的研究方向，期待藉由了解體外培養唾液腺細胞的生長歷程與離體細胞培養環境的調控，可以取得更有利於植入細胞的培養天期與條件，對於日後發展唾液腺組織工程運用在臨床病人身上有所裨益。

Salivary gland is an exocrine gland that is responsible for saliva production. The secretion of saliva contains digestive enzymes, growth factors, and antimicrobial agents. This function is crucial in the processes of digestion, lubrication, and protection in the body. Therefore, salivary gland hypofunction can result serious negative effects on the patients quality of life. Radiation therapy (RT) for head and neck cancer, and autoimmune diseases (such as Sjogren’s syndrome) can cause unavoidable coirradiation of surrounding normal tissues such as the salivary glands. This effect results in irreversible damage of salivary glands hence to significantly reduces salivary output. Salivary gland hypofunction and xerostomia lead to hindering of speech (dysphonia), difficulty of swallowing (dysphagia), influences on nutrition (dysnutritia) and others. Currently, there is no treatment available to permanently treat dysfunctional salivary glands. Constructing artificial salivary glands with tissue engineering may be a viable curative procedure to treat patients suffering from dry mouth. Tissue engineering applications in salivary glands require a significant amount of salivary gland cells and an understanding of gland cell-related coordination and function. Many experimental induce the increase of salivary gland stem/progenitor cells with duct ligation or fluorescence-activated cell sorting by antibodies. This research establishes a simple and highly reproducible protocol for isolation and characterization of stem/progenitor cells obtained from human salivary glands. The percentage of CD49f and CD90 double-positive cells was increased at 14 days after seeding, and then decreased at 28 days. The gene expression levels of ALDH1A1 and ALDH1A3 were also at the highest at 14 days after seeding. Cultured cells can successfully differentiate into adipocytes and osteocytes. E-cadherin expression in cultured cells increased with time while vimentin expression gradually decreased to a very low level after prolonged culture, inferring a MET during the repopulation process of cell culture. Our study confirmed the existence of progenitor cells in cultured adult human parotid glands. This research was also established a simple culture method to obtain sufficient progenitor cells for further tissue engineering studies. However, maintaining the expression levels of amylase in culturing salivary gland epithelial cells is important.Salivary gland cells in vivo are surrounded by a complex stromal environment, in which fibroblasts are the main cell type in proximity to the gland cells. An appropriate number of fibroblasts in contact with the parotid gland acinar cells (PGACs) is necessary to promote PGAC function. Fibroblast-secreted bFGF may play a paracrine signaling role in the regulation of α-amylase expression in PGACs. Interestingly, the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced. By growth factor protein array assay, higher NT-4 expression was observed in PVDF-derived fibroblast conditioned medium. Fibroblasts might be reprogrammed into neural-like cells after cultured on appropriate biomaterials. In these studies, the interaction between PGACs, fibroblasts, and biomaterials was investigated. This could be treated patients with xerostomia and applying directly by re-implantation of autologous salivary gland tissue engineering. The re-implantation treatment can be a major contribution to their quality of life.