臺灣博碩士論文加值系統

自西元1982年之後，惡性腫瘤便成為台灣十大死因之首位。肺癌更高居台灣地區男性及女性癌症死亡率的第二位以及第一位。許多研究顯示肺癌的發生與暴露環境中的致癌物有關。而吸煙率偏低的華人女性卻有偏高的肺癌發生率，其重要危險因子有待探討。Interleukin-17（IL-17）是一種前發炎趨物，主要是由活化的CD4＋T細胞所分泌，可以誘發各種細胞產生IL-6和IL-8；並且已被證明在鼠類的模式中是一種血管生成和腫瘤促進因子。IL-6和IL-8已被指出與肺癌的致病機轉有關；另外transforming growth factor-β1（TGF-β1）和cyclooxygenase-2（COX-2）也參與癌症的生成，因此我們想要研究IL-17在人類肺癌細胞中可否調控這些細胞激素的產生。我們加入IL-17於七株肺癌細胞株中，利用RT-PCR方法觀察IL-6、IL-8、TGF-β1和COX-2基因的表現以及利用ELISA方法測定IL-6、IL-8和TGF-β1蛋白的表現量。實驗結果顯示在肺癌細胞株中幾乎所有細胞株經過IL-17刺激後，IL-6和IL-8皆有被誘發的現象，尤其以CH27細胞最為明顯；因此，進一步以免疫組織染色方法來探討IL-17在肺癌組織中的蛋白表現情形以及其與臨床分類上之相關性，結果發現IL-17主要表現於腫瘤細胞周圍的淋巴球以及壞死區和肺腺癌細胞中，但並不表現於肺鱗狀癌細胞中。接下來的研究欲評估IL-17是否可當做肺癌病人預後的指標，於是我們抽取肺癌病人化療前及化療後之血清利用ELISA方式檢測其IL-17的含量，結果發現健康成人、肺癌病人以及經過化療後的病人血清中的IL-17含量並無差別。本研究的最終目的欲評估IL-17是否會促進肺腫瘤的增生及轉移，目前已將IL-17的cDNA送入pCDNA的載體中，進一步欲將這些含IL-17基因的載體轉染至肺癌的細胞株中，再把這些肺癌細胞株注射入裸鼠內觀察其腫瘤增生及轉移的情形。

Malignant neoplasms have been the primary cause of death in Taiwan since 1982. Lung cancer is the leading and the second-leading cause of cancer deaths among women and men, respectively, in Taiwan. Exposure to air pollutants is considered as a causal factor in the development of lung cancer. However, the prevalence of cigarette smoking is low among Chinese females. The high mortality among Chinese females was unexplained by the habit of cigarette smoking. The important risk factors needed to be investigated. Interleukin (IL)-17 is a proinflammatory cytokine which is secreted mainly by activated human memory CD4＋ T cells that induces IL-6 and IL-8 from various cells. It has been demonstrated that IL-17 is an angiogenic factor and can promote tumor growth in murine tumor model. IL-6 and IL-8 have been implicated in the pathogenesis of lung cancer. The transforming growth factor（TGF-β1）and cyclooxygenase（COX-2） also involve in tumorigenicity. Therefore, in this study we want to investigate how IL-17 regulates these cytokines in lung cancer cells. We showed that in vitro, IL-17 can increase IL-6、IL-8、TGF-β1 and COX-2 expression in lung cancer cell lines at both protein and mRNA levels. The enhancement of cytokine expression is obvious, especially for the CH27 cell line. Thereafter, we used immunohistochemistry（IHC）method to study the expression of IL-17 in lung cancer tissues and relationship between IL-17 expression and clinicopathologic parameters. We found that the expression of IL-17 focuses in lymphocytes surrounded the necrosis area. Also, the expression of IL-17 can be found in adenocarcinoma lung cancer tissues but not in squamous lung cancer tissues. Continuously we would like to know if IL-17 could be used as a prognosis factor for lung cancer patients. The sera were came from normal healthy adults and lung cancer patients before and after receiving chemotherapy. The IL-17 levels from sera were detected by ELISA method. The levels of IL-17 were not different from lung cancer patients and normal adults. The final aim of this research is to study if IL-17 could promote tumor growth and metastasis. We have already inserted the IL-17 cDNA into a pCDNA vector, the next step will transfect this pCDNA vector containing IL-17 gene into lung cancer cell lines and inject these lung cancer cell lines containing IL-17 gene into nude mice to investigate the role of IL-17 in tumorigenicity and metastasis.

目 錄
中文摘要 1
英文摘要 3
第一章 前言
第一節 肺癌流行病學及危險因子 5
第二節 肺癌分類及其預後 7
第三節 Interleukin-17與細胞激素之關係 9
第四節 研究動機 20
第二章 材料與方法
第一節 材料
一、 材料與藥品 22
二、人類肺癌細胞株來源 22
三、檢體來源 22
第二節 方法
一、細胞培養、分盤 25
二、RNA純化與RT-PCR 26
三、ELISA測定 28
四、免疫組織染色法 29
五、免疫組織染色結果之判讀 31
六、 統計分析 31
七、 淋巴球的製備 31
八、 Interleukin-17產物的製備 32
九、 由agarose gel回收 DNA片段（Elution） 33
十、 PCR產物的接合反應（Ligation reaction） 34
十一、 轉型作用（Transformation） 35
十二、 質體DNA抽取 36
第三章 實驗結果
第一節 Recombinant interleukin-17在肺癌細胞株中對細胞激素基因之影響 38
第二節 Recombinant interleukin-17在肺癌細胞株中對細胞激素蛋白之影響 42
第三節 Interleukin-17在肺癌病人組織中之表現情形 45
第四節 肺癌病人血清中Interleukin-17 之表現情形 47
第五節 構築（construct）可以產生重組（recombinate）的interleukin-17蛋白 48
第四章 討論 49
第五章 圖表目錄
圖一 在H1355肺癌細胞株中，不同的濃度的IL-17與處理時間對於IL-6基因的影響 54
圖二 在H1355肺癌細胞株中，不同濃度的IL-17與處理時間對於IL-6 蛋白的影響 55
圖三 處理IL-17對不同肺癌細胞株之IL-6基因表現的影響 56
圖四 處理IL-17 對不同肺癌細胞株之IL-8基因表現的影響 57
圖五 處理IL-17 對不同肺癌細胞株之COX-2基因表現的影響 58
圖六 處理IL-17 對不同肺癌細胞株之TGF-β1基因表現的影響 59
圖七 正常人肺組織切片中，並沒有發現 IL-17表達的情形 60
圖八 肺腺癌組織切片 60
圖九 腫瘤壞死區IL-17陽性淋巴球表現的情形 61
圖十 正常人及肺癌病人化療前後血清中的IL-17的測定 62
圖十一 構築（construct）可表達IL-17的質體流程圖 63
圖十二 IL-17質體限制作用之電泳凝膠分析圖 64
表一 處理IL-17對肺癌細胞株IL-6蛋白表現的影響 65
表二 處理IL-17對肺癌細胞株IL-8蛋白表現的影響 66
表三 處理IL-17對肺癌細胞株TGF-β1蛋白表現的影響 67
表四 肺癌病人腫瘤之臨床表徵 68
表五 Interleukin-17在非肺癌組織及肺癌腫瘤組織中淋巴球的表現情形 70
表六 Interleukin-17在不同臨床表徵的肺癌組織中之表現情形 71
表七 Interleukin-17 於肺癌組織中的表現量與臨床指標之相關性 73
附表一 RT-PCR引子（primer）序列 75
附表二 各種引子（primer）的PCR條件 76
參考文獻 77
目 錄
中文摘要 1
英文摘要 3
第一章 前言
第一節 肺癌流行病學及危險因子 5
第二節 肺癌分類及其預後 7
第三節 Interleukin-17與細胞激素之關係 9
第四節 研究動機 20
第二章 材料與方法
第一節 材料
一、 材料與藥品 22
二、人類肺癌細胞株來源 22
三、檢體來源 22
第二節 方法
一、細胞培養、分盤 25
二、RNA純化與RT-PCR 26
三、ELISA測定 28
四、免疫組織染色法 29
五、免疫組織染色結果之判讀 31
六、 統計分析 31
七、 淋巴球的製備 31
八、 Interleukin-17產物的製備 32
九、 由agarose gel回收 DNA片段（Elution） 33
十、 PCR產物的接合反應（Ligation reaction） 34
十一、 轉型作用（Transformation） 35
十二、 質體DNA抽取 36
第三章 實驗結果
第一節 Recombinant interleukin-17在肺癌細胞株中對細胞激素基因之影響 38
第二節 Recombinant interleukin-17在肺癌細胞株中對細胞激素蛋白之影響 42
第三節 Interleukin-17在肺癌病人組織中之表現情形 45
第四節 肺癌病人血清中Interleukin-17 之表現情形 47
第五節 構築（construct）可以產生重組（recombinate）的interleukin-17蛋白 48
第四章 討論 49
第五章 圖表目錄
圖一 在H1355肺癌細胞株中，不同的濃度的IL-17與處理時間對於IL-6基因的影響 54
圖二 在H1355肺癌細胞株中，不同濃度的IL-17與處理時間對於IL-6 蛋白的影響 55
圖三 處理IL-17對不同肺癌細胞株之IL-6基因表現的影響 56
圖四 處理IL-17 對不同肺癌細胞株之IL-8基因表現的影響 57
圖五 處理IL-17 對不同肺癌細胞株之COX-2基因表現的影響 58
圖六 處理IL-17 對不同肺癌細胞株之TGF-β1基因表現的影響 59
圖七 正常人肺組織切片中，並沒有發現 IL-17表達的情形 60
圖八 肺腺癌組織切片 60
圖九 腫瘤壞死區IL-17陽性淋巴球表現的情形 61
圖十 正常人及肺癌病人化療前後血清中的IL-17的測定 62
圖十一 構築（construct）可表達IL-17的質體流程圖 63
圖十二 IL-17質體限制作用之電泳凝膠分析圖 64
表一 處理IL-17對肺癌細胞株IL-6蛋白表現的影響 65
表二 處理IL-17對肺癌細胞株IL-8蛋白表現的影響 66
表三 處理IL-17對肺癌細胞株TGF-β1蛋白表現的影響 67
表四 肺癌病人腫瘤之臨床表徵 68
表五 Interleukin-17在非肺癌組織及肺癌腫瘤組織中淋巴球的表現情形 70
表六 Interleukin-17在不同臨床表徵的肺癌組織中之表現情形 71
表七 Interleukin-17 於肺癌組織中的表現量與臨床指標之相關性 73
附表一 RT-PCR引子（primer）序列 75
附表二 各種引子（primer）的PCR條件 76
參考文獻 77
目 錄
中文摘要 1
英文摘要 3
第一章 前言
第一節 肺癌流行病學及危險因子 5
第二節 肺癌分類及其預後 7
第三節 Interleukin-17與細胞激素之關係 9
第四節 研究動機 20
第二章 材料與方法
第一節 材料
一、 材料與藥品 22
二、人類肺癌細胞株來源 22
三、檢體來源 22
第二節 方法
一、細胞培養、分盤 25
二、RNA純化與RT-PCR 26
三、ELISA測定 28
四、免疫組織染色法 29
五、免疫組織染色結果之判讀 31
六、 統計分析 31
七、 淋巴球的製備 31
八、 Interleukin-17產物的製備 32
九、 由agarose gel回收 DNA片段（Elution） 33
十、 PCR產物的接合反應（Ligation reaction） 34
十一、 轉型作用（Transformation） 35
十二、 質體DNA抽取 36
第三章 實驗結果
第一節 Recombinant interleukin-17在肺癌細胞株中對細胞激素基因之影響 38
第二節 Recombinant interleukin-17在肺癌細胞株中對細胞激素蛋白之影響 42
第三節 Interleukin-17在肺癌病人組織中之表現情形 45
第四節 肺癌病人血清中Interleukin-17 之表現情形 47
第五節 構築（construct）可以產生重組（recombinate）的interleukin-17蛋白 48
第四章 討論 49
第五章 圖表目錄
圖一 在H1355肺癌細胞株中，不同的濃度的IL-17與處理時間對於IL-6基因的影響 54
圖二 在H1355肺癌細胞株中，不同濃度的IL-17與處理時間對於IL-6 蛋白的影響 55
圖三 處理IL-17對不同肺癌細胞株之IL-6基因表現的影響 56
圖四 處理IL-17 對不同肺癌細胞株之IL-8基因表現的影響 57
圖五 處理IL-17 對不同肺癌細胞株之COX-2基因表現的影響 58
圖六 處理IL-17 對不同肺癌細胞株之TGF-β1基因表現的影響 59
圖七 正常人肺組織切片中，並沒有發現 IL-17表達的情形 60
圖八 肺腺癌組織切片 60
圖九 腫瘤壞死區IL-17陽性淋巴球表現的情形 61
圖十 正常人及肺癌病人化療前後血清中的IL-17的測定 62
圖十一 構築（construct）可表達IL-17的質體流程圖 63
圖十二 IL-17質體限制作用之電泳凝膠分析圖 64
表一 處理IL-17對肺癌細胞株IL-6蛋白表現的影響 65
表二 處理IL-17對肺癌細胞株IL-8蛋白表現的影響 66
表三 處理IL-17對肺癌細胞株TGF-β1蛋白表現的影響 67
表四 肺癌病人腫瘤之臨床表徵 68
表五 Interleukin-17在非肺癌組織及肺癌腫瘤組織中淋巴球的表現情形 70
表六 Interleukin-17在不同臨床表徵的肺癌組織中之表現情形 71
表七 Interleukin-17 於肺癌組織中的表現量與臨床指標之相關性 73
附表一 RT-PCR引子（primer）序列 75
附表二 各種引子（primer）的PCR條件 76
參考文獻 77

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