林中天臺灣大學：獸醫學研究所駱虹霏Lo, Hung-FeiHung-FeiLo2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/60002蛋白質酪胺酸磷酸化之訊息傳遞在許多不同種類的細胞中，已被證實在細胞增生、細胞與細胞間的接合、細胞移動和細胞骨骼的構成中，扮演著極為重要的角色，但目前僅有非常有限的研究著重於此一訊息傳遞對角膜內皮細胞的影響。本篇研究的目的為想要了解酪胺酸磷酸化之訊息傳遞在角膜內皮細胞與細胞之間接合所扮演之調控角色。我們推測酪胺酸磷酸酶 (PTP)在角膜內皮細胞與細胞間的接合扮演著極重要的角色，一旦使用酪胺酸磷酸酶的抑制劑 (PTP inhibitor)來抑制其功能，就會使細胞與細胞間的接合瓦解，並誘發許多下游的反應使得細胞進行分裂和改變其通透性。實驗材料為初代培養之牛角膜內皮細胞和全厚度之兔子眼角膜，使用sodium orthovanadate (SOV)作為酪胺酸磷酸脢的抑制劑，接著以不同SOV濃度 (25, 50, 100μM)及不同時間 (8, 24 hrs)來處理初代培養之細胞與新鮮的兔子眼角膜，之後進行螢光染色並使用螢光顯微鏡及共軛焦顯微鏡進行影像擷取，以觀察位於細胞與細胞間的蛋白質，例如：N-cadherin, alpha-catenin 和p120的改變，另外也使用Ki67抗體來偵測是否有進入cell cycle的細胞。細胞間接合蛋白質和一些cell cycle的調節蛋白質 (例如：Cyclin A, Cyclin E, Cyclin D1和PCNA)的表現量則利用西方墨點法 (Western blotting)來定量。實驗結果顯示酪胺酸磷酸脢的抑制劑 (PTP inhibitor)會改變角膜內皮細胞的細胞形態、打斷原本細胞與細胞間的緊密接合，也會使原本一直停留在G1 phase早期的細胞重新進入細胞週期 (cell cycle)；但是本實驗中所選定的若干細胞間接合蛋白質和cell cycle的調節蛋白質其表現量卻沒有改變。未來的研究期望能更瞭解磷酸化之訊息傳遞如何影響角膜內皮細胞特性的機制。The importance of phosphotyrosine signal transduction in cellular proliferation, cell-cell contact and cellular migration has been proved in various cell types. However, only limited studies have been reported on corneal endothelial cell cells. In this study, we aim to understand the role of phosphotyrosine signaling in corneal endothelial cellular function. We propose that protein tyrosine phosphotase (PTP) may play an important role in cell-cell junction of corneal endothelial cells, and the disruption of its function by PTP inhibitor can break through cell-cell junction, and trigger a lot of downstream actions such as cellular proliferation and change of permeability. Primary culture of bovine corneal endothelial cells and whole rabbit corneas were used as the experimental materials. We first treated the cultured bovine corneal endothelial cells with PTPs inhibitor, sodium orthovanadate (SOV), with a variety of concentrations (25,50,100μM) for various durations (8,24 hrs). The effects of PTP inhibition on cellular distribution of cell-cell junctional proteins, such as N-cadherin, alpha-catenin and p120, were evaluated by immunohistochemical staining with fluorescein microscopy and confocal microscopy. Immunohistochemical staining with Ki67 Ab, a marker of cell proliferation, was also used to detect cells entering cell cycle. The expression levels of these cellular junctional proteins and regulatory proteins in cell cycle regulation such as Cyclin A, Cyclin E, Cyclin D1 and PCNA, were quantified by Western blotting. Our results demonstrate that PTPs inhibitor broke through cell-cell junction, and triggered corneal endothelial cells to re-enter cell cycle instead of having no impacts on celluar proliferation. However, the expression levels of those chosen cell-cell junction proteins and chosen regulatory proteins in cell cycle regulation were unchanged. Further studies will be carried out to elucidate the mechanism of phosphotyrosine signaling in mediating the cellular behavior of corneal endothelial cells.摘要……………………………………………………………….…….…1 Abstract…………………………………………………………………..3 第一章 序言……………………………………………..……….…4 第二章 文獻探討 第一節 角膜內皮細胞與其生理功能之簡介………………………8 第二節 細胞接合 (cell-cell junctions)與細胞間接合蛋白質 (junctional protein)之簡介……………………………11 I. Tight junctions (TJs)………………………………….12 II. Adherens junctions (AJs)………………………………12 第三節 酪胺酸磷酸化之訊息傳遞 (phosphotyrosine signal transduction)對細胞生理之重要性………………………15 第四節 細胞生長循環 (cell cycle)調控系統之簡介…………...17 第五節 接觸抑制（contact inhibition）與角膜內皮細胞 分裂的相關實驗之文獻探討……………………….………19 第三章 實驗材料與方法 第一節 牛角膜內皮細胞之初代培養………………………………..22 第二節 Junctional protein 於細胞內之表現分析 I. 細胞免疫化學染色 (Immunocytochemistry)……………..23 II. 組織免疫螢光染色 (Immunofluorescence)………………23 III. 西方墨點法 (Western blotting)…………………………24 第三節 Sodium orthovanadate (SOV)之製備與使用……………....27 第四章 實驗結果 第一節 接合蛋白質細胞免疫化學染色於培養之牛角膜內皮細 胞之表現……………………………………………………..29 第二節 接合蛋白質 (junctional protein)之表現分析…………36 第三節 Ki-67於培養之牛角膜內皮細胞之表現……………………39 第四節 細胞生長循環相關蛋白質 (regulatory proteins in cell cycle regulation)之表現分析………………………43 第五節 接合蛋白質p120組織免疫化學染色於新鮮之兔眼角膜 內皮細胞之表現…………………………………….………..45 第五章 討論 一. 人類角膜內皮細胞在體內無法分裂的原因...………...…………48 二. 角膜細胞培養與組織培養之使用與比較…………………..51 三. PTPs inhibitor 對細胞間接合蛋白質 (junctional protein)之影響…………………………………………….52 四. PTPs inhibitor對內皮細胞分裂之影響…………………53 五. 本實驗之限制……………………………………………….54 六. 未來實驗方向及其臨床應用性…………………………….55 第六章 參考文獻………………………………………….………..563260463 bytesapplication/pdfen-US角膜酪胺酸磷酸化內皮細胞訊息傳遞磷酸化corneaPTPsendotheliumsignal transductionphosphotyrosinethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60002/1/ntu-94-R91629027-1.pdf