臺灣博碩士論文加值系統

在台灣，肺癌死亡率占惡性腫瘤中的首位，並有逐年增加的趨勢。由於肺癌在女性中發生率漸增且大多數女性並沒有吸菸的習慣，因此雌激素 (17β-estradiol, E2)被推測於肺癌的病理機轉具有重要的地位。另外，有研究指出，E2可以促使骨橋蛋白 (osteopontin, OPN)的表現，而OPN與腫瘤的增生、移行、侵入及轉移有關。因此，我們擬探討E2及OPN在肺癌細胞的成長及細胞移行所扮演的角色，並釐清其分子訊息路徑。此外，合併使用tamoxifen citrate及gefitinib是否能對肺癌細胞生長及移行產生更有效的抑制也是我們觀察的目標。首先，我們在A549細胞株發現E2會促使細胞生長、移行以及活化基質金屬蛋白酶 (matrix metalloproteinase, MMP-2)；在PE089肺腺癌細胞株也同樣觀察到E2會引發細胞移行。而使用MEK抑制劑U0126則會抑制細胞移行顯示E2的作用是透過活化MEK/ERK訊息傳遞路徑。合併使用tamoxifen citrate及gefitinib兩種藥物處理細胞株後，發現可以更有效的抑制細胞的移行能力。在A549細胞株，E2可以促進OPN RNA的表現，而tamoxifen citrate則可以抑制此現象。此外，我們收集來自12個癌症病人肋膜積水檢體，偵測其OPN及E2的濃度。結果發現，在lung adenocarcinoma病人，肋膜積水中OPN及E2的濃度高於控制組3倍，而且OPN與E2兩者的濃度具有正相關性。以肋膜積水培養A549及PE089細胞株。結果發現，在A549細胞株，細胞移行的能力與OPN或E2並沒有正相關性；而在PE089細胞株中，細胞移行的能力與OPN有正相關性，但與E2則沒有關連。綜合實驗結果，我們推論未來臨床上合併使用tamoxifen citrate及gefitinib可以思考做為難治肺癌的一種治療方式，另外，OPN可能可以作為一個肺癌治療預測因子及預後的指標。

Lung cancer is the most common cause of cancer death in Taiwan. The incidence of lung cancer is increasing in female and most of them don’t smoke. Estrogen (E2) is proposed to play an important role in pathogenesis of lung cancer. Besides, previous studies reported estrogen could stimulate osteopontin (OPN) expression, and OPN has an important role in tumor proliferation, migration, invasion and metastasis. We try to explore the roles of estrogen and OPN in lung cancer cell lines growth and migration, and investigate the participated signaling pathway. The separate and combined effect of tamoxifen citrate and gefitinib on the tumor cell growth and migration was also studied. We found estrogen could stimulate A549 lung cancer cell line growth, migration and activation of MMP-2. In PE089 lung cancer cell line, estrogen could stimulate cell migration. The effect was through the activation of MEK/ERK signaling pathway. Synergistic effect of tamoxifen citrate and gefitinib on the inhibition of cell migration was noted. Estrogen could stimulate OPN RNA expression in A549 cell line, and tamoxifen citrate antagonized the effect. We collected pleural effusions from twelve cancer patients and measured their OPN and E2 concentrations. The pleural fluid OPN and E2 concentrations from patients with lung adenocarcinoma are three times more than the control. We tried adding the pleural fluid to cultured A549 and PE089 cell lines There is no correlation between pleural fluid E2 concentrations and migration in both cell lines. But, positive correlation between pleural fluid OPN concentration and migration was found in PE089 cell line. According to the above results, gefitinib combined with tamoxifen citrate may be a new treatment modality for patients with refractory lung cancer and OPN could be looked as a predictor for treatment and prognosticator.

中文摘要 1
Abstract 3
研究動機及目標 5
第一章 文獻回顧 6
肺癌 (Lung cancer) 7
肺癌種類 7
肺癌成因 8
治療方式 10
雌激素 (Estrogen) 11
雌激素合成 11
雌激素功能 12
雌激素受體(estrogen receptor, ER) 12
雌激素受體的分子結構 12
雌激素受體的分布及功能 13
雌激素受體與肺癌的相關研究 14
骨橋蛋白 (Osteopontin) 15
第二章 實驗材料與方法 17
藥品試劑 18
實驗方法 20
一、人類肺腺癌細胞培養 20
二、檢體收集 20
三、藥物配置 21
四、細胞計數 21
五、分析細胞內RNA表現 22
六、細胞蛋白質製備(Preparation of cell lysate) 23
七、粒線體分離 (Mitochondrial fraction) 24
八、蛋白質定量法(Protein assay) 25
九、西方墨點法(Western blotting) 25
十、Scratch wound assay 27
十一、Gel zymography 27
十二、肋膜積水之骨橋蛋白(OPN)的測定 29
十三、雷射共軛顯微鏡 30
十四、統計分析 31
第三章 實驗結果與分析 32
一、 偵測肺癌細胞株中是否含有ER 33
二、 雌激素 (E2)對於肺癌細胞株生長曲線 (growth curve)、存活率(viability)的影響 33
三、 E2對於肺癌細胞株移行能力的影響 34
四、 E2對於肺癌細胞株表現MMP-2的影響 34
五、 E2藉由活化MEK-ERK訊息傳遞路徑調控細胞移行能力 35
六、 合併臨床藥物tamoxifen citrate及gefitinib對於肺癌細胞的影響 36
七、 E2對於肺癌細胞株之OPN的表現的影響 37
八、 E2透過刺激OPN表現以促進細胞移行能力 37
九、 OPN對於肺癌細胞株移行能力的影響 38
十、 偵測肋膜積水 (Pleural effusion)中OPN及E2的含量 38
十一、 肋膜積水促進細胞移行能力與OPN及E2含量的相關性 39
十二、 觀察ERβ在肺腺癌細胞中的分布情形 40
第四章 討論 41
第五章 參考文獻 51
第六章 圖表 59
第七章 附件 84

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