慢性阻塞性肺病、特發性肺纖維化、肺癌發生皆與吸菸行為高度相關。其中慢性阻塞性肺病與肺癌更是高居2012年世界十大死因之一。先前研究指出上皮-間質轉化 (EMT) 可能涉及這些肺部疾病，但相關機制目前尚未完全了解。EMT 是指上皮細胞失去其原有特徵，逐漸獲得間質細胞特徵之生物過程，主要發生在胚胎形成、組織修復、器官纖維化、腫瘤進展與轉移之過程中，與上述肺部疾病發生有重要關係。因此，研究香菸煙造成之肺細胞上皮-間質轉化現象有助於進一步了解這些肺部疾病以及治療方法。
我們測試香菸萃取物 (CSE) 對人類肺上皮A549細胞株之影響，發現CSE會造成A549細胞死亡，而且會隨著CSE暴露時間與濃度增加而變嚴重。另一方面，沒有被CSE殺死之細胞會呈現一些EMT特徵。這些特徵包括型態延長、表面失去頂纖毛、移動力增加、E-cadherin表達減少、Vimentin和α-smooth muscle actin表達增加。最近研究指出間葉幹細胞有治療肺部疾病之潛力，而且可能是藉由旁分泌(Paracrine) 之作用產生療效。因此，我們將A549細胞培養於脂肪間葉幹細胞培養液 (ADSC-CM) ，發現ADSC-CM對CSE造成之細胞死亡有保護作用。此外，我們發現ADSC-CM能減緩CSE誘導細胞EMT特徵之效果。這些結果說明細胞EMT會受生存環境影響，在有害生存之環境中細胞EMT特徵比較明顯；在受保護之環境，例如含有ADSC-CM之環境中，EMT特徵就不明顯。最後，我們利用基因微陣列分析EMT相關基因表現，經CSE暴露後，發現A549細胞的上皮細胞特徵基因表達減少，少數間質細胞特徵基因表達增加。因此在本研究中，A549細胞只進行「部分EMT」。我們的研究結果也證明未來可以利用基因微陣列分析進一步確認類似EMT之細胞現象。
整體而言，我們呈現CSE對A549細胞之影響，包括細胞死亡和EMT。此外，我們藉由不同的影像分析方法詳細討論A549細胞類似EMT之特徵。最後，我們利用ADSC-CM減緩CSE造成之A549細胞死亡與EMT，表示在這些吸菸行為相關之肺部疾病中，脂肪間葉幹細胞提供了旁分泌治療之可能性。

Chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer are deadly disease related to smoking. COPD and lung cancer were among the 10 leading causes of death in the world in 2012. Previous studies suggest that epithelial-mesenchymal transition (EMT) may play a role in COPD, IPF and lung cancer; however, the mechanism linking EMT to these dieases remains incompletely understood. EMT describes a biological process by which epithelial cells lose their epithelial characteristics and gradually acquire mesenchymal traits, and this process mainly occurs in embryogenesis, tissue regeneration, organ fibrosis, cancer progression and metastasis. The causes of lung diseases mentioned above are highly associated with fibrosis, cancer progression and metastasis. Therefore, study on cigarette smoke-induced EMT in lung epithelial cells contributes to understanding and better treatment for these diseases.
We investigated the effect of cigarette smoke extract (CSE) on human lung epithelial A549 cell line in vitro. Our results demonstrated that CSE induced A549 cell death in a concentration- and time-dependent manner. In addition, the cells escaped from CSE-induced cell death exhibited some EMT properties: elongated shape, lack of apical microvilli, increased motility, decreased E-cadherin expression, and increased Vimentin and -smooth muscle actin expression. Recent studies suggest the therapeutic potential of mesenchymal stem cells for lung diseases, which is probably mediated by paracrine actions of mesenchymal stem cells. Accordingly, A549 cells were cultured with adipose-derived stem cell condition medium (ADSC-CM), and we found that ADSC-CM had a protective effect on CSE-induced cell death; moreover, ADSC-CM repressed CSE-indued EMT properties in A549 cells. These results imply that microenvironment plays an important role in EMT induction. A549 cells tended to undergo EMT in adverse condition, but CSE induced EMT properties in A549 cells became less evident in a less adverse condition such as with the inclusion of ADSC-CM. Finally, we investigated EMT related genes by microarray analysis. After CSE exposure, we found that genes as epithelial markers were downregulated in A549 cells, but only few genes as mesenchymal markers were upregualted. As a result, A549 cells underwent “partial EMT” by CSE induction, and we show that such EMT-like phenomena can be further confirmed by microarray analysis.
In summary, we demonstrated the effect of CSE on A549 cells, including cell death and EMT. Furthermore, we discussed EMT-like features in A549 cells via different methods for image analysis. Finally, we used ADSC-CM to repress CSE-indued cell death and EMT in A549 cells, which suggest that ADSCs have therapeutic potential for smoking related lung diseases by paracrine actions.

摘要 i
Abstract ii
誌謝 iii
目次 iv
圖目次 vii
表目次 ix
第1章 緒論 1
1.1 文獻回顧 1
1.1.1 吸菸引起肺部疾病 1
1.1.2 上皮-間質轉化 4
1.1.3 間葉幹細胞治療肺部疾病 10
1.2 研究動機與目的 12
第2章 材料與方法 14
2.1 抗體 (Antibodies) 14
2.2 藥品與溶液 (Chemicals and Solutions) 14
2.3 細胞培養 (Cell culture) 17
2.4 製備細胞培養液 (Preparation of condition medium) 17
2.5 細胞增生分析 (Cell proliferation assay) 18
2.6 細胞毒性分析 (Cytotoxicity assay) 19
2.7 上皮-間質轉化之誘導 (Induction of EMT) 21
2.8 光學顯微鏡觀察 (Light microscopy) 21
2.9 掃描式電子顯微鏡觀察 (Scanning electron microscopy) 22
2.9.1 細胞培養與固定 (Cell culture and Fixation) 22
2.9.2 脫水與臨界點乾燥 (Dehydration and Critical point drying) 23
2.9.3 離子覆膜 (Ion sputter coating) 24
2.9.4 掃描式電子顯微鏡觀察 (Scanning electron microscopy) 25
2.10 傷口癒合分析 (Wound healing assay) 26
2.11 免疫螢光染色 (Immunofluorescence staining) 27
2.12 西方墨點法 (Western Blot) 31
2.12.1 細胞培養與萃取 (Cell culture and extraction) 31
2.12.2 蛋白質定量 (Quantitation of protein) 31
2.12.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis，SDS-PAGE) 32
2.12.4 西方墨點法 (Western blot) 33
2.12.5 條帶密度分析 (Densitometric analysis of bands) 35
2.13 基因微陣列分析 (Microarray analysis) 35
第3章 實驗結果 38
3.1 香菸萃取物影響A549細胞之生長 38
3.1.1 香菸萃取物抑制細胞增生及降低細胞存活率 38
3.1.2 香菸萃取物造成之細胞毒性 45
3.2 香菸萃取物誘導A549細胞發生上皮-間質轉化 50
3.2.1 細胞型態與表面結構改變 50
3.2.2 細胞移動力增加 55
3.2.3 上皮-間質轉化相關標記蛋白改變 59
3.3 脂肪間葉幹細胞培養液減緩香菸萃取物對A549細胞之影響 66
3.3.1 脂肪間葉幹細胞培養液對香菸萃取物造成之細胞死亡有保護作用 66
3.3.2 脂肪間葉幹細胞培養液減緩香菸萃取物誘導之A549細胞上皮-間質轉化 75
3.4 基因微陣列分析 91
第4章 討論與結論 99
4.1 細胞死亡 99
4.2 上皮-間質轉化 102
4.3 基因表現 107
4.4 結論 117
參考文獻 119
附錄一 139
附錄二 143
附錄三 148
附錄四 149

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