臺灣博碩士論文加值系統

前言：角膜組織在正常情況下不存在淋巴管和血管，創傷會引起的角膜完整性破壞、以及纖維母細胞的活化，可能引起新生血管、纖維化和瘢痕形成，導致視力損害。轉化生長因子-β（TGF-β）在角膜上皮損傷後通過促進纖維母細胞形成，α-SMA由纖維母細胞誘導活化， Smads蛋白已被證明是由TGF-β超家族成員特異性活化，並調節細胞生長、增殖、遷移和分化。高碳酸血症是血液中二氧化碳濃度異常升高的情況，過去的研究已經知道高碳酸血症酸中毒對許多器官都具有保護作用。目前還沒有有效的局部治療方法可以治癒角膜瘢痕，所以預防角膜疤痕的形成至關重要。
研究目的：以體外細胞模式，了解高碳酸血症酸中毒對於TGF-β1誘導的角膜纖維母細胞纖維化之影響及可能的機轉，以期提供一個能抑制角膜纖維化的策略。
材料與方法：使用人類角膜纖維母細胞(Corneal Fibroblast, CFs)以TGF-β1誘導角膜損傷，並給予不同時間的CO2通氣，分別為控制組(5% CO2)、HCA組 (10% 1小時)、TGF-β1組(TGF-β1 10 ng/ml、5% CO2)、TGF-β1+HCA 1小時組(TGF-β1 10 ng/ml、10% CO2 1小時)、TGF-β1+HCA 2小時組(TGF-β1 10 ng/ml、10% CO2 2小時)、以及TGF-β1+HCA 3小時組(TGF-β1 10 ng/ml、10% CO2 3小時) ，使用西方墨點法、細胞免疫螢光染色、細胞遷移試驗等方式進行研究。
研究結果：HCA處理可以藉由降低不論是經典途徑或是替代途徑之NF-κB信號路徑的活化，以及抑制TGF-β/Smad訊號路徑的活化達到抑制纖維化發生的情形，並且HCA處理可以降低TGF-β1誘導的角膜纖維母細胞的遷移作用、細胞入侵作用、以及對膠原凝膠的收縮情形。
結論：HCA可以透過抑制IKK-NF-κB以及TGF-β/Smad訊號傳遞路徑的活化來減少角膜纖維母細胞的纖維化情形，並達到抑制細胞遷移的作用。

Background: The corneal tissue does not have lymphatic vessels and blood vessels under normal conditions, the destruction of corneal integrity caused by trauma, and the activation of fibroblasts may cause neovascularization, fibrosis and scar formation, resulting in visual impairment. Transforming growth factor beta (TGF-β) promotes fibroblast formation after corneal epithelial damage, and α-SMA is activated by fibroblasts. Smads have been shown to be specifically activated by TGF-β superfamily members and regulate cell growth and proliferation. , migration and differentiation. Hypercapnia is an abnormally elevated concentration of carbon dioxide in the blood, and past studies have known that hypercapnia acidosis has protective effects on many organs. At present, there is no effective local treatment to cure corneal scars, so it is important to prevent the formation of corneal scars.
Objective: To understand the effects of hypercapnic acidosis on TGF-β1-induced fibrosis of corneal fibroblasts and elucidate possible mechanisms for a strategy of anti-corneal fibrosis.
Materials and Methods: Corneal injury was induced by TGF-β1 using human corneal fibroblasts (CFs), and CO2 ventilation was given at different times, which were control group (5% CO2), HCA group (10% 1 hour), TGF-β1 group (TGF-β1 10 ng/ml, 5% CO2), TGF-β1+HCA 1 hour group (TGF-β1 10 ng/ml, 10% CO2 1 hour), TGF-β1+HCA 2 hour group (TGF-β1 10 ng/ml, 10% CO2 for 2 hours), and TGF-β1+HCA for 3 hour group (TGF-β1 10 ng/ml, 10% CO2 for 3 hours), using Western blotting, cell immunofluorescence staining, cell migration experiments and other methods were conducted.
Main Results: HCA treatment inhibited TGF-β1 induced corneal fibroblast migration, cell invasion, and contraction of collagen gels. The TGF-β1 induced activation of the NF-κB signaling pathway, both classical pathway and the alternative pathway, and Smad signaling pathway were suppressed by HCA treatment.
Conclusions: HCA can reduce TGF-β1 induced corneal fibroblast fibrosis by inhibiting IKK-NF-κB and Smad signaling pathways. It could be a potential therapy for corneal fibrosis.

第一章、緒言 1
第一節、 角膜纖維母細胞 1
壹、 角膜 1
貳、 角膜輪部 2
參、 角膜緣纖維母細胞 2
第二節、 轉化生長因子-β1 4
第三節、 核轉錄因子-κB 6
壹、 經典NF-κB路徑 7
貳、 替代NF-κB路徑 7
第四節、 TGF-β與NF-κB信號傳導路徑 8
第五節、 高碳酸血症 8
壹、 高碳酸血症酸中毒 8
貳、 高碳酸血症的保護機制 10
第六節、 研究目的 11
第二章、材料與方法 12
第一節、 實驗材料及設備 12
壹、 藥品及試劑 12
貳、 儀器 12
參、 細胞培養 12
肆、 細胞冷凍保存 13
伍、 細胞解凍 14
陸、 細胞計數 14
第二節、 實驗流程及組別 15
壹、 實驗流程 15
貳、 實驗組別 15
第三節、 細胞遷移試驗 16
壹、 細胞刮傷實驗 16
貳、 細胞入侵 17
參、 膠原蛋白膠收縮 17
第四節、 細胞免疫螢光染色 18
第五節、 西方墨點法 19
壹、 蛋白質萃取 19
貳、 蛋白質濃度測定 20
參、 蛋白質轉漬 20
第六節、 統計分析 21
第三章、結果 23
第一節、 HCA處理對TGF-β1誘導角膜纖維母細胞膠原凝膠收縮影響23
第二節、 HCA處理對TGF-β1誘導角膜纖維母細胞的遷移影響 24
壹、 細胞刮傷實驗 24
貳、 細胞入侵 25
第三節、 HCA處理對TGF-β1誘導角膜纖維母細胞纖維化的影響 25
第四節、 HCA處理對TGF-β1誘導角膜纖維母細胞NF-κB信號路徑的影響 26
壹、 經典途徑 26
貳、 替代途徑 27
參、 NF-κB在核內的表現 27
第五節、 HCA處理對TGF-β1誘導角膜纖維母細胞SMAD信號路徑的影響 28
第四章、討論 41
第一節、 HCA與NF-κB的關係 41
第二節、 HCA與角膜傷口癒合的關係 42
第三節、 高碳酸血症與酸中毒 42
第四節、 未來展望 44
第五章、結論 46
第六章、參考文獻 47

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