臺灣博碩士論文加值系統

放射線治療是頭頸部癌症患者的重要治療方法，而位在頭頸部的唾液腺體，對輻射敏感度特別的高，因此在接受放射線治療後的病患，往往會出現發炎以及口唇乾燥的現象 (Xerostomia)。雖然此現象終極原因為唾液腺的被破壞，但其分子機制尚未完全了解，但已有部分資料顯示，口乾症狀極可能與唾液腺細胞膜上的水通道蛋白(Aquaporins)有所關聯，其中特別又以Aquaporin5 (AQP5)最為重要。人體內有各種水通道蛋白，隨著組織不同表現不一樣的水通道蛋白，此蛋白有選擇性通透的功能，不僅能有效運送水分並且可以隔絕離子的進出。AQP5主要表現在眼睛、肺部與唾液腺體，在此蛋白運作下，使得唾液腺體順利把水分排出，因此本實驗將探討輻射傷害是否會影響AQP5在唾液細胞中的表現。我們使用人類唾液腺體細胞株A253進行實驗，在不同劑量的輻射照射後，藉此觀察細胞生存率並比對AQP5的表現；另外將大鼠進行頭頸部的輻射照射，依不同時間點收集唾液流量，最後再研究AQP5表現量是否改變，證明唾液流量的減少並非腺體細胞凋亡所致，而是因為AQP5表現量降低所造成，研究結果顯示，經輻射照射後的大鼠，單位體重在時間內之唾液分泌量確實降低，而唾液腺體之AQP5表現量也下降，希望此結果有助於了解唾液分泌量減少的機制，以利未來能有效減緩輻射所引起的口乾症現象。

Radiotherapy is a major treatment in the management of head and neck cancer, and the salivary glands are very sensitivity to irradiation. The patients with cancers over head and neck received radiation-therapy might result in severe damage on salivary glands. This complication induced inflammation and loss of function on salivary glands. The patients received irradiation would feel dry mouth (xerostomia) resulting from lack of saliva and sometimes the treatment program will be cancelled due to the complication. Although the pathogenesis of this disease is still not very clear, some researches indicated the dysfunction of aquaporins (AQPs), especially AQP5, may play a vital role on xerostomia.
AQPs are a family of homologous membrane proteins that function to control highly selective water channels. There are many kinds of AQPs in our body and AQP5 is mainly distributed over salivary glands, such as parotid, submandibular, and sublingual glands. Therefore, AQP5 should be very important for salivary secretion. The aims of this study are trying to figure out the pathogenesis and molecular mechanism of AQP5 on radiation induced xerostomia and to evaluate the effect of irradiation on apoptosis and change of expression of AQP5.
First, we used human salivary gland cell line, A253, to measure the relationship between expression levels of AQP5 irradiation. Second, we performed animal study using SD rats to expose to radiation, then measure the secreting and saliva and collect the salivary glands for analyses. According to our preliminary results indicated that the decoration of saliva was decreased and the expression of AQP5 was reduced after irradiation. We attempted to determine whether the radiation-induced inflammatory reaction diminishing the expression of AQP5 but not apoptosis and we hope we can find out a strategy to protect the function of salivary gland exposed to irradiation.

第一章 緒論
第一節、輻射引起的口乾症狀 (Radiation induced Xerostomia) …1
第二節、唾液的分泌 (Salivary secretion)…………………………3
第三節、Aquaporins & Aquaporin 5………………………………4
第四節、Aquaporin 5傳導路徑…………………………...……… 6
第五節、Pilocarpine………………………………………………. 6
第六節、研究動機與方向…………………………………………7
第七節、研究策略…………………………………………………8

第二章 材料與方法
第一節、細胞培養（Cell culture）…………………………………9
第二節、細胞存活與細胞增生試驗 (MTT assay)
（Cell survival and tetrazolium dye colorimetric assay）… 9
第三節、細胞週期與細胞死亡試驗 (PI stain )
(Analysis of cell death and cell cycle) ………………….11
第四節、質體的抽取與製備
(Preparation of recombinant plasmids) …………………12
第五節、DNA接合反應 （DNA ligation）………………………14
第六節、細胞轉染 （Transfection）………………………………15
第七節、動物實驗（Animals model）………………….……… 16
第八節、免疫螢光染色 (Immunofluorescence)…………………17
第九節、常規染色 (H&E stain) …………………………………17
第十節、收集唾液淨流量 (Saliva collection)……………………18
第十一節、RNA抽取與半定量PCR (Semi-quantitative RT-PCR)19
第十二節、抽取細胞株蛋白質 (Protein extraction)…………… 20
第十三節、西方墨點法（Western blotting）……………………21
第十四節、統計分析 (Statistical analytics)……………………...25

第三章 結果
第一節、於A253細胞株Aquaporin的表現…………………….26
第二節、Pilocarpine藥物在A253細胞株的作用……………….27
第三節、利用MTT方式測量A253細胞株的存活率………… 27
第四節、利用PI方式測量A253細胞株的細胞週期與死亡率28
第五節、驗證A253在接受不同輻射劑量後 AQP5表現的變
化………………………………………………………..29
第六節、測試Aquaporin於SD大鼠唾液腺的表現情形….…...30
第七節、經輻射照射後大鼠唾液流量的變化………………..….30
第八節、輻射照射引起大鼠體重與腺體重量的改變情況….….31
第九節、唾液流量與腺體細胞數量的關聯性………………..…31
第十節、輻射照射40天後大鼠唾液腺體AQP5的表現量下降…32
第四章 討論……………………………………………………………33

第五章 參考文獻………………………………………………………38

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