臺灣博碩士論文加值系統

幾丁聚醣(chitosan)具有促進上皮細胞間的緊密連接(tight junction, TJs)打開的功能，使藥物進入細胞間隙，增加藥物吸收，因此被廣泛運用於口服親水藥物或鼻腔給藥劑型之藥物載體。許多文獻證實，幾丁聚醣具有打開TJs的能力，但幾丁聚醣打開TJs的機制，及對細胞內分子層面的影響與變化，仍未詳細了解。因此本研究分成兩個部分探討：第一部分為探討幾丁聚醣打開TJs，是否與細胞表面膜蛋白間的相互作用有關。用trypsin將表面膜蛋白切除後，再將幾丁聚醣加入細胞。發現細胞在TEER、西方墨點法、細胞間隙穿透試驗以及免疫螢光染色分析的結果都顯示，細胞膜蛋白切除後，幾丁聚醣打開TJs的能力會下降。第二部分進一步探討幾丁聚醣打開TJs時，可能活化的訊息傳遞路徑。利用Protein array分析，篩選出與幾丁聚醣作用相關的酪胺酸激酶（tyrosine kinase）。結果發現，當幾丁聚醣打開TJs時，訊息傳遞路徑上的PKCJ、FAK及Src酪胺酸激酶會發生磷酸化。經由文獻探討，回溯這條磷酸化路徑找出可能的受體蛋白 (receptor)為 integrin ?庮??。上述實驗，均以claudin-4是否瓦解，當作TJs打開的依據。本實驗結果初步證實，幾丁聚醣打開TJs，可能是經由活化細胞表面的integrin受體蛋白，使下游的酪胺酸激酶磷酸化，造成TJs上的claudin-4蛋白瓦解，而打開TJs。

摘要 I
誌謝 II
目錄 II
表目錄 III
圖目錄 IV
第一章 緒論 1
1. 前言 1
1.1 幾丁聚醣(chitosan) 2
1.2 緊密連接(Tight junction) 3
1.2.1 Tight junction結構及組成 4
1.2.2 Tight Junction功能 6
1.3 幾丁聚醣對TJs的影響 6
訊息傳遞路徑 7
1.4 7
1.4.1 受體蛋白(Receptor) 7
1.4.2 訊息傳遞路徑（Signaling Pathway） 8
1.4.3 Integrin and Src kinase activation 10
1.5 研究動機與目的 11
第二章 幾丁聚醣打開TJs與細胞表面蛋白質之關聯 14
2.1 研究目的 14
2.2 材料與方法 14
2.2.1 細胞培養 14
2.3 Tight junction通透度測試 16
2.3.1 培養Caco-2單層細胞 16
2.3.2 配製幾丁聚醣溶液 19
2.3.3 配製Thermolysin 及Trypsin溶液 19
2.3.4 膜蛋白切除實驗與膜蛋白染色 19
2.4 蛋白質的表現 21
2.4.1 細胞膜蛋白及質蛋白質之萃取 21
2.4.2 西方墨點法(Western blot) 22
2.4.3 免疫螢光染色 23
2.5.1 Trypsin 切除細胞表面膜蛋白 25
2.5.2 Trypsin 造成TJs的破壞 26
2.5.3 Thermolysin對Trypsin阻斷 27
2.5.4 幾丁幾醣對去除表面膜蛋白細胞TJs的影響 31
2.6 結果討論 34
第三章 幾丁聚醣造成的磷酸化活化路徑與表面受體的關聯 35
3.1研究目的 35
3.2 材料與方法 35
3.2.1 蛋白質表現 35
3.2.2 訊息傳遞路徑 40
3.2.3 表面受體 41
3.2.4 幾丁聚醣與表面受體作用 42
3.3.1 受體蛋白種類選取 44
3.3.2 Protein array 篩選相關Tyrosine kinase磷酸化蛋白 46
3.3.3 西方墨點法確認相關路徑磷酸化蛋白 47
3.3.4 抑制劑作用 49
3.3.5 與幾丁聚醣作用的表面受體及電腦模擬 52
3.4 結果與討論 57
參考文獻 58

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