臺灣博碩士論文加值系統

根據衛生署公佈的國人死因統計資料指出，癌症早已高居台灣十大死因之首位，而絕大多數之病人都是直接或間接死於癌細胞的遠處轉移。癌細胞的轉移是一個複雜的過程，為一個或一群癌細胞由原組織脫落，藉由血液或淋巴循環系統散佈到其他組織或器官的血管中，並穿過血管壁於其落地生根增殖。而腫瘤細胞會分泌胞外基質分解酵素來分解胞外基質 (extracellular matrix) 及血管壁，將細胞帶入或帶離血管，所以破壞與分解基底膜和胞外基質是癌細胞進行侵犯轉移時所必要的過程。
目前，國內外研究癌病發現，惡性腫瘤細胞會產生的胞外基質分解酶包括有serine proteinase、matrix metalloproteinases (MMP)、cathepsins、plasminogen activator及heparanitase等。其中MMP在基底膜的破壞扮演極重要的角色，與癌症的侵犯與轉移有著密切的關係。MMP是一群會分解細胞間質之蛋白酶，目前在人體中已發現超過21種MMP亞型，可分解不同類型的胞外基質。相關研究顯示MMP對於腫瘤細胞的生長、侵犯和轉移有著重要的相關性。MMP的專一抑制劑為Tissue inhibitor of matrix metalloproteinase (TIMP)，為最主要的MMP的內生性調控者，可專一抑制MMPs的活性，在癌症的發展過程中，MMP與TIMP之間的平衡關係是相當重要的。
胸腔腫瘤的發病率和死亡率有逐年上升的趨勢，台灣地區的危險度較他國偏高，衛生署所公佈的民國95年國人癌症死因統計中更明列肺癌為國人癌症死因排行榜中之榜首（男性居第二、女性居首位）。由於肺癌癒後差，通常診斷時且已屆晚期；而胸腔另一常見的腫瘤-食道癌近年來發生率亦急遽升高，在2006年台灣的發病率亦名列前十名中，（第九名）。先前我們實驗室進行了在非小細胞肺癌的骨髓微侵犯 (microinvolvement) 的研究，分析非小細胞肺癌病患的骨髓微侵犯的現象，而所謂的骨髓微侵犯的現象就是在骨髓中偵測到腫瘤細胞。我們發現超過30%的非小細胞肺癌病患及超過40％的食道癌病患有骨髓微侵犯的現象。雖然骨髓微侵犯與病人的年齡、性別、細胞型態與TNM分期確認沒有相關性，然隨後更進一步的實驗我們卻發現表現MMP-13的非小細胞肺癌腫瘤細胞較易在骨髓中聚集，且其5年累積存活率亦較差。
為了能夠更進一步瞭解MMP家族在腫瘤細胞轉移時所扮演的角色，此次的研究主要將在細胞培養基中執行。利用不同的細胞株，我們將分析不同培養基濃度、結構、生長因子對細胞生長的影響。隨後將使用最適切的細胞株及生長條件來觀察MMP2、MMP9對腫瘤細胞生長及移行能力之影響。

According to the annual report of Department of Health, Executive Yuan, ROC（Taiwan）, cancer has already became the leading cause of the ten major deaths in Taiwan . Lung cancer is the number one cause of cancer death in Taiwan, that it about 70% lung cancer patients whose death was caused by cancer cells metastasis. Tumor metastasis is a complex process involving coordination of matrix disintegration, cell detachment, cell-cell junction disassembly, and cell migration. The process is facilitated by the tumor cells’ ability to degrade their surrounding extracellular matrix (ECM), especially basement membranes lining the blood vessels. Increased degradation of ECM and basement membranes through secretion of ECM-degrading enzymes enables the cells to enter and leave blood vessels. Degradation of basement membranes and ECM is required for tumor cell invasion and metastasis.
The incidence of disease and death rate of the thoracic tumor rise sharply, danger degree of Taiwan is on the high more side than that of other country. The lung cancer is in compatriot's cancer ranks, man occupy second, women and rank first. The prognosis situation of lung cancer is usually bad, due to diagnosed at a late stag. Another common tumor of thorax with rapid increase of incidence in recent years is esophageal cancer to ten malignancy in 2006’s report . The extracellular matrix proteinase secreted by malignant cancer cell, including serine proteinase, matrix metalloproteinases (MMP), cathepsins, plasminogen activator, and heparanitase. The MMPs are the key enzymes in the degradation of basement membranes and extracellular matrix. MMPs are a group of cellular matrix proteinase. In human, the MMP family consists of at least 21 different members, which can degrade different type of ECM. The relation between MMP and tumor cell growth, invasion, and metastasis is directly proportional. Proteolytic activity of MMP is inhibited by specific inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMP). Under cancer develop the expression of MMP and TIMP is highly coordinated.
Our previous study using immunohistochemical stain has demonstrated occurrence of bone-marrow microinvolvement in more than 30% of the non-small cell lung cancer patients, and more than 40％ of the esophageal cancer patients, respectively. Detected a single tumor cell in bone-marrow is call bone-marrow microinvolvement. Though the occurrence of bone-marrow microinvolvement was not related to patient age, sex, cell type, or TNM stages, subsequent study from our lab demonstrated that existence of BMM itself did not influence the prognosis (p=0.109), however, patients with positive MMP-13 expression had a poorer 5-year survival rate of 26.5% (p=0.025). These data indicate that non-small cell lung cancer cells with MMP-13 expression, despite of BMM status, tend to shed and aggregate in the bone marrow, which is subsequently reflected in a poorer survival rate. The current study will be conducted in the cell culture medium. Using different cell lines, we will analyze the impact of structure and concentration of medium, and growth factor on cell growth. By selecting the most suitable cell lines and growth condition, then we will analyze the expression of MMPs on tumor growth and migration.

目錄
誌謝 I
中文摘要 VI
Abstract VIII
第一章 前言 1
1.1 細胞的介紹 1
1.2 細胞的生長因子 2
1.2.1 PDGF 3
1.2.2 EGF 3
1.3 癌症的形成 4
1.4 癌症的治療 4
1.5 肺癌 5
1.5.1 肺癌的診斷及治療流程 6
1.5.2 非小細胞肺癌 6
1.5.3 小細胞肺癌 6
1.6 食道癌 6
1.6.1 鱗狀上皮細胞癌 7
1.6.2 腺癌 8
1.7 膠原蛋白 8
1.8 MMP Family 10
第二章 緒論 13
第三章 材料與方法 15
3.1 細胞株 15
3.2 藥品 15
3.3 儀器 19
3.4 方法 19
3.4.1蛋白質濃度分析 19
3.4.2蛋白質電泳 (Protein electrophoresis) SDS-PAGE 20
3.4.3細胞數目之測定 21
3.4.4Gelatin zymography 22
3.4.5細胞移行分析 chemotaxis assay 22
3.4.6細胞侵襲分析 invasion assay 23
3.4.7本實驗之培養基配置 23
第四章 實驗結果 24
4.1 細胞的篩選 24
4.2 三維培養基之建立 24
4.3 生長因子的角色 25
4.4 腫瘤細胞的移行及侵犯 25
4.4.1生長因子的角色 26
4.4.2 MMPs的角色 26
4.5 生長因子與MMPs表現之關係 27
第五章 討論 29
第六章 結論 32
第七章 參考文獻 References 33
第八章 附圖表 39
表一. 膠原蛋白的型式及組成 39
表二. 基體金屬蛋白酶的分類 (MMPs Family) 40
表三. 不同生長因子對細胞的調控 41
表四. 細胞種類中英對照表 42
圖一. 細胞在一般環境中的細胞數目表現 (In medium) 43
圖二. 細胞在處理生長因子PDGF後的細胞數目表現 (In medium) 44
圖三. 細胞在處理生長因子EGF後的細胞數目表現 (In medium) 45
圖四. 細胞在一般環境中的細胞數目表現 (In collagen) 46
圖五. 細胞在處理生長因子PDGF後的細胞侵犯表現 ( Boyden chamber) 47
圖六. 細胞在處理生長因子EGF後的細胞侵犯表現 ( Boyden chamber) 48
圖七. 細胞在侵犯試驗中的細胞數目表現（上層為S.F medium；下層為collagen）49
圖八. 細胞在侵犯試驗中的百分比表現（上層為S.F medium；下層為collagen）50
圖九. 細胞在侵犯試驗中的細胞數目表現（上層為S.F medium；下層為collagen）51
圖十. 各種類細胞在細胞盤培養盤中第三天的影像 （100x） 52
圖十一. 細胞在細胞培養基中第七天的影像 （200x） 53
圖十二. Boyden Chamber簡易示意圖 54
圖十三. 細胞在未經處理後移行16小時後的影像 （100x） 55
圖十四. 細胞在處理PDGF後侵犯試驗中12小時後的影像（100x） 56
圖十五. 細胞在處理EGF後侵犯試驗中12小時後的影像 （100x） 57
圖十六. 細胞在培養基中加入生長因子EGF萃取其中之MMP做Gelatin Zymography 58
圖十七. 細胞在培養基中萃取其中之MMP做Gelatin Zymography 59
圖十八. 細胞在培養基中加入生長因子PDGF萃取其中之MMP做Gelatin Zymography 60
圖十九. 細胞在培養基中萃取其中之MMP做Gelatin Zymography 61
圖二十. 細胞在培養基中萃取其中之MMP做Gelatin Zymography 62

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