- 學位論文
經鹵素衍生物處理之腮腺纖維母細胞對唾液腺細胞分泌唾液澱粉酶之影響
Effect of halide treated-parotid fibroblasts on α-amylase release of parotid gland acinar cells
摘要
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，可能由於老化或自體免疫引起的唾液腺發炎，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。 欲使組織工程成功重建唾液腺體，分泌細胞與間質細胞之間的交互作用值得探討。研究指出，利用生醫材料為基底培養身為間質細胞的纖維母細胞，會分泌出一神經營養因子Neurotriphin 4 (NT4)影響分泌細胞釋出唾液澱粉酶的功能。當中效果最好的生醫材料為聚偏氟乙烯(polyvinylidene fluoride, PVDF)。由於PVDF為含氟側枝的單純高分子，因此認為鹵素族化合物會對唾液腺功能有促進的效果。 本研究將纖維母細胞與分泌細胞自組織中分離培養，三週後待細胞爬出並穩定後將纖維母細胞培養在含有鹵素的高分子膜材或者外加含鹵素鹽類的培養基中三天。後收取其培養液並培養分泌細胞。藉此了解高分子型態以及離子型態的鹵素化合物對刺激纖維母細胞分泌生長因子誘導分泌細胞產出唾液澱粉酶的效率如何。經由免疫螢光染色、西方點墨法，以及核酸序列定量偵測等分析方式觀察分泌細胞之唾液澱粉酶之產生與胞外分泌量。 其中在高分子組別除了已知的PVDF有良好的效果外，聚氯乙烯(polyvinyl chloride, PVC)亦有較佳的表現在唾液澱粉酶的分泌上。經PVDF以及PVC培養的纖維母細胞皆有分泌較多的NT4。而離子組別中僅經氟化鈉(sodium fluoride， NaF)處理之分泌細胞製造出較多的唾液澱粉酶。然而檢測其纖維母細胞之NT4的量並無顯著表現，故推測細胞經離子刺激後所循的途徑與高分子不同，即鹵素離子使纖維母細胞影響分泌細胞的方式不是依靠NT4。 藉由離體培養了解如何利用外在刺激使分泌細胞周邊的間質細胞自主分泌生長因子調控分泌唾液澱粉酶，掌握調控分泌細胞功能之方法後即可往立體培養之方向前進。從結果得知PVDF以及PVC可藉由刺激NT4釋放使分泌細胞表現卓越，而NaF亦然。但是就目前研究發現兩者誘導分泌細胞製造唾液澱粉酶的途徑並不同，故期望將NaF與PVC或者PVDF結合共同刺激纖維母細胞會產生協同效應，增加效率。以此期待可以使口乾症患者治療更簡易且增加組織工程之效率。
並列摘要
Salivary gland is an exocrine gland that is responsible for saliva production. The secretion of saliva contains digestive enzymes, growth factors, and antimicrobial agents. Saliva helps to swallow and digest food bolus, and it can clean oral cavity by flushing the food debris away. As a result, patients who suffered salivary gland hypofunction will affect their life quality. Aging, 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. The leakage in saliva secretion leads to xerostomia, and the following side effects are hindering of speech (dysphonia), difficulty of swallowing (dysphagia), influences on nutrition (dysnutritia) and others. However, there are no effective treatments to cure the damage after irradiation for head neck cancer currently. Therefore, constructing artificial salivary glands with tissue engineering may be a viable curative procedure to treat patients suffering from dry mouth. It is important to realize the interaction between mesenchymal cells and parotid gland acinar cells (PGACs) before reconstructing salivary glands. A previous study said that utilize biomedical material to culture parotid gland fibroblasts, part of mesenchymal cells, and they released a specific growth factor. Such growth factor is a part of neurotrophic factors called neurotrophin 4 (NT4). Stimulated by NT4, PGACs released much α-amylase. Among these biomedical materials, polyvinylidene fluoride (PVDF) induced fibroblasts secreting more amounts of NT4. Because the structure of PVDF is simple, the reasons of fibroblasts induction may attribute to halogen family. In this study, we cultured fibroblast and PGACs from tissue respectively for 21 days, and then seeded fibroblasts on different halide polymers or in medium containing halogen ions for three days. The conditioned medium collected from fibroblasts then cultured PGACs for another three days. Immunofluorescence staining, western blot, and QPCR were conducted to investigate the efficiency of α-amylase production from PGACs. Besides different halogen, we also discussed that whether polymer form or ionic form was more sufficient. Except for PVDF, polyvinyl chloride (PVC) performed better in regulating α-amylase secretion as well. Amounts of NT4 in PVC and PVDF-derived fibroblasts were high, however, it was mediocre in NaF-derived fibroblasts which induced PGACs releasing much more α-amylase as compared to other halogen ion. As a consequence, we considered that the way halogen ion influence fibroblasts regulating PGACs doesn’t depend on NT4. After realizing how to stimulate mesenchymal cells secreting growth factor by external stimulus, and then influence functions of PGACs. The next stage is to develop 3D culture. Based on our results, combination of NaF and PVC/ PVDF will cause synergistic effects because the pathways polymer and ion follow are different. Therefore, we expect such combination could improve efficiency of tissue engineering and make the treatments more convenient.