臺灣博碩士論文加值系統

肺部纖維化 (pulmonary fibrosis) 是一個漸進式的慢性疾病，會產生乾咳、易喘、呼吸困難的症狀，嚴重時還可能合併肺高壓及心肺衰竭，死亡率很高。而肺部纖維化的過程大多是肺部細胞受到傷害後，為了癒合而產生許多細胞激素及蛋白質，而不正常的細胞訊息傳遞路徑則會導致像是肺部纖維母細胞等細胞增生、膠質分泌量增加，取代原本的肺泡細胞並使其失去功能。環境干擾物 (Environmental endocrine disruptor) 是存在於日常生活中，在一定的濃度下會影響人體正常生理功能的化學分子，本研究主要目的在探討環境干擾物對於肺部纖維化的的影響以及其相關細胞訊息傳遞路線。由西方點墨法(Western blot)和免疫螢光染色及共軛焦顯微鏡的結果，發現環境干擾物的刺激會增加肺部纖維母細胞表現分化的特定蛋白??-SMA，而其中2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)的刺激會增加Cycloxygenase-2(COX-2)的表現量和芳香烴受體 (Aryl Hydrocarbon Receptor) 進入細胞核，由半定量PCR及定量PCR的結果看出，戴奧辛增加了纖維母細胞的CYP1B1基因表現量，從細胞移動分析中發現環境干擾物刺激後，細胞移動能力有顯著的增加，接著又以單細胞鈣離子影像分析 (Calcium thermal image)發現以戴奧辛刺激後的短時間內，細胞內鈣離子濃度瞬間增加。而在以siRNA抑制芳香烴受體的表現之後，發現??-SMA及cytosolic phospholipase A2 (cPLA2)表現下降， COX-2表現量上升。因此，根據以上實驗結果，我們推論戴奧辛除了經由芳香烴受體去影響基因層面的細胞訊息傳遞之外，還可能透過非基因路徑(Non-genomic pathway)去調控其他的訊息傳遞路徑，影響肺部纖維母細胞的分化、移動、蛋白質表現及發炎反應的產生，進而有可能影響肺部纖維母細胞的分化及肺部纖維化的產生。

Pulmonary fibrosis is a progressive process that leads to cough, shortening of breath, pulmonary hypertension and cardiopulmonary failure. Fibrosis is defined by the overgrowth, scarring of tissues and is attributed to excess deposition of extracellular matrix including collagen and fibronectin, leading to reduced pulmonary function. Environmental endocrine disruptors are compounds that interfere with human endocrine system and have been shown to be assotiated with many diseases. Therefore, the purpose of our study was to investigate the effects of environmental endocrine disruptors on pulmonary fibroblasts and the signaling transduction pathway involved. We found that environmental endocrine disruptors increased the ??-SMA protein expression in two lung fibroblast cell lines. The cell migration ability was increased by the treatment of the cells with environmental endocrine disruptors. TCDD also induced COX-2 protein expression and Aryl hydrocarbon receptor (AhR) nuclear localization. The expression of CYP1B1 gene confirmed the activation of AhR signaling pathway induced by TCDD. Calcium thermal imaging of the cells indicated that TCDD in-creased the intracellular calcium concentration in short-term treatment. Western bloting analysis showed that cytosolic phos-pholipase A2 (cPLA2) was induced after treatment of the cells with TCDD. The expression of ??-SMA and cPLA2 was reduced, while the expression of COX-2 increased in cells with AhR knockdown. According to these results, we suggested that TCDD induced fibroblast differentiation, migration, ??-SMA protein expression and cPLA2 protein expression through AhR signaling pathway. Therefore, TCDD induced the cal-cium-dependent inflammatory pathway may exacerbate lung fibroblasts differentiation and pulmonary fibrosis.

中文摘要…………………………………………………………………2英文摘要…………………………………………………………………4致謝………………………………………………………………………6
目錄………………………………………………………………………8圖表目錄………………………………………………………………12
第一章 前言…………………………………………………………14
第一節 肺部纖維化………………………………………………14
1-1 簡介………………………………………………………14
1-2 肺部纖維化的病理現象與治療…………………………14
1-3 肺部纖維化與慢性發炎的關係…………………………15
第二節 環境干擾物………………………………………………16
2-1 簡介………………………………………………………16
2-2 鄰苯二甲酸酯類…………………………………………17
2-3 戴奧辛……………………………………………………17
第三節 環境干擾物誘導的細胞訊息傳遞路徑…………………18
3-1 芳香烴受體之細胞訊息傳遞路徑………………………18
3-2 細胞內鈣離子訊息傳遞路徑……………………………19
第二章 研究目的……………………………………………………20
第三章 實驗設計……………………………………………………21
第一節 環境干擾物對肺部纖維母細胞的影響…………………21
3-1a環境干擾物刺激肺部纖維母細胞的??-SMA蛋白質表現
3-1b環境干擾物刺激肺部纖維母細胞的細胞移動情形
3-1c 環境干擾物刺激肺部纖維母細胞的膠原蛋白分泌情形
3-1d 環境干擾物刺激肺部纖維母細胞的基質金屬蛋白水解酶
活性表現
3-1e 環境干擾物刺激肺部纖維母細胞的細胞生長情形
3-1f 環境干擾物刺激肺部纖維母細胞後的基因表現情形
第二節 環境干擾物誘發的訊息傳遞路徑之探討………………23
3-2a 環境干擾物刺激肺部纖維母細胞後Smad3活化情形
3-2b 芳香烴受體在環境干擾物刺激肺部纖維母細胞後的入核
情形
3-2c 環境干擾物刺激肺部纖維母細胞後CYP1B1表現情形
3-2d 環境干擾物刺激肺部纖維母細胞的COX-2蛋白質表現
3-2e 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
芳香烴受體蛋白質表現
3-2f 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
??-SMA蛋白質表現
3-2g 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
COX-2蛋白質表現
第三節 鈣離子在環境干擾物誘發的訊息傳遞路徑扮演的角色25
3-3a 環境干擾物刺激肺部纖維母細胞的細胞內鈣離子變化
3-3b 環境干擾物刺激肺部纖維母細胞的cPLA2蛋白質表現
3-3c 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
cPLA2蛋白質表現
第四章 實驗材料與方法……………………………………………27
第五章 實驗結果……………………………………………………36
第一節 環境干擾物對肺部纖維母細胞的影響…………………36
5-1a環境干擾物刺激肺部纖維母細胞的??-SMA蛋白質表現
5-1b環境干擾物刺激肺部纖維母細胞的細胞移動分析
5-1c 環境干擾物刺激肺部纖維母細胞的膠原蛋白分泌情形
5-1d 環境干擾物刺激肺部纖維母細胞的基質金屬蛋白水解酶
活性表現
5-1e 環境干擾物刺激肺部纖維母細胞的細胞生長情形
5-1f 環境干擾物刺激肺部纖維母細胞後的發炎相關基因表現
第二節 環境干擾物誘發的訊息傳遞路徑之探討………………39
5-2a 環境干擾物刺激肺部纖維母細胞後Smad3活化情形
5-2b 芳香烴受體在環境干擾物刺激肺部纖維母細胞後的入核
情形
5-2c 環境干擾物刺激肺部纖維母細胞後CYP1B1表現情形
5-2d 環境干擾物刺激肺部纖維母細胞的COX-2蛋白質表現
5-2e 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
芳香烴受體蛋白質表現
5-2f 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
??-SMA蛋白質表現
5-2g 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
COX-2蛋白質表現
第三節 鈣離子在環境干擾物誘發的訊息傳遞路徑扮演的角色44
5-3a 環境干擾物刺激肺部纖維母細胞的細胞內鈣離子變化
5-3b 環境干擾物刺激肺部纖維母細胞的cPLA2蛋白質表現
5-3c 抑制芳香烴受體後環境干擾物刺激肺部纖維母細胞的
cPLA2蛋白質表現
第六章 討論…………………………………………………………46
第一節 環境干擾物對於人欸肺部纖維母細胞分化與結構改變之
影響………………………………………………………46
第二節 環境干擾物誘導的細胞訊息傳遞路徑之探討…………49
圖表……………………………………………………………………52
參考文獻………………………………………………………………88

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