臺灣博碩士論文加值系統

Desmocollin-2(DSC2)基因是desmosome的成員之一，desmosome是一個跨膜蛋白在細胞的粘附中扮演重要的角色。最近的研究指出在大腸結腸癌、乳癌和鱗狀食道癌中發現DSC2表現降低，顯示DSC2表現減少可能參與癌症的發展。在實驗室以前的研究結果顯示DSC2基因的表現與肺癌細胞的轉移及侵入能力呈現負相關。我們發現在低轉移的細胞株抑制DSC2基因表現會促進肺癌細胞的移動能力，反之在高轉移的細胞株大量表現DSC2會抑制肺癌細胞的移動能力。為了探討DSC2基因所影響的細胞訊息傳遞以及分子調節的作用機制。我們將抑制DSC2基因的細胞株進行微陣列技術，發現NDRG1基因可能受到DSC2基因的影響。有研究指出NDRG1基因表現會促進癌症細胞的轉移和入侵，而且我們在低轉移細胞株大量表現NDRG1基因也看到細胞的移動能力增加，因此在大量表現DSC2的細胞株中重新表現NDRG1，確實NDRG1增強了原本受到DSC2抑制細胞的移動能力，最後我們的推斷DSC2基因可能藉由NDRG1基因調控細胞的轉移和入侵。

Desmocollin-2 (DSC2) associated with cancer metastasis. DSC2 gene is one of the desmosome family, desmosome is a transmembrane protein that plays an important role in cell adhesion. Reduced expression of DSC2 has been reported in colorectal cancer, breast cancer and oesophageal squamous cell carcinoma. Suggesting that DSC2 may play a role in the development and progression of cancer. In preliminary results, we found that DSC2 gene expression levels in lung cancer cells were negatively correlation with the invasion activity of a panel of lung cancer cell lines. We suggested that knockdown DSC2 gene expression promote lung cancer cells migration and invasion in low metastasis cell CL1-0. On the contrary, overexpression DSC2 gene suppressed lung cancer cells migration and invasion in high metastasis cell CL1-5. To investigate the possible DSC2 gene signal transduction and molecular regulation. We were proceeded microarray assay of shDSC2 gene lung cancer cell line. We found that DSC2 gene possible regulates NDRG1 gene. Many studies reported that NDRG1 gene promote lung cancer cell migration and invasion. Overexpression NDRG1 gene increase lung cancer cell migration and invasion in low metastasis cell CL1-0. Moreover, restoration of NDRG1 expression in DSC2-transfected cells abrogated DSC2-induced anti-invasion activity. Together, our results indicate that DSC2 inhibits human lung cancer cell migration and invasion by suppressting NDRG1 gene expression.

封面內頁
簽名頁
中文摘要 iii
英文摘要 iv
誌謝 v
目錄 vi
圖目錄 x

1. 前言 1
1.1 癌症與癌轉移 1
1.2 Desmosome的結構與癌症 2
1.3 Desmocollin-2 (DSC2)與癌症 4
1.4 DSC2和NDRG1的相關性 5
1.5 N-myc downstream regulated gene 1 (NDRG1)的結構與功能 6
1.6 NDRG1在癌症中的關係 8
2. 研究動機 10
3. 實驗設計與流程 11
4. 材料與方法 13
4.1 肺癌細胞株 13
4.2 細胞繼代培養 13
4.3 細胞凍存 14
4.4 質體DNA萃取 15
4.4.1 細菌培養和細菌凍存 15
4.4.2 萃取質體DNA 15
4.5 DNA電泳 16
4.6 建立穩定轉染之細胞株 17
4.6.1 轉染(Transfection) 17
4.6.2 細胞連續稀釋挑選stable clone 17
4.7 RNA萃取 18
4.8 cDNA合成 19
4.9 即時定量PCR 19
4.10 西方墨點法 20
4.10.1 SDS膠體配置 20
4.10.2 蛋白質之定量曲線 21
4.10.3 細胞蛋白質萃取 22
4.10.4 SDS膠體電泳 23
4.10.5 半乾式電轉 23
4.10.6 抗體雜合和使用照膜儀器呈現結果 24
4.11 細胞遷移能力分析(wound healing assay) 24
4.12 細胞入侵能力分析(transwell invasion assay) 25
4.13 細胞與細胞之黏附分析 (cell-cell adhesion assay) 25
4.13.1 細胞懸滴分析 (hanging drop assay) 26
4.14 MTT分析 26
4.15 細胞群落形成分析 (colony formation assay) 27
4.17 非貼附之細胞群落形成分析 (soft agar assay) 27
4.18 David functional annotation tool 28
5. 結果 29
5.1 建立抑制DSC2基因之肺癌細胞株 29
5.2 抑制DSC2基因會促進肺癌細胞的遷移和入侵能力 29
5.3 抑制DSC2基因會減少肺癌細胞與細胞間的黏附能力 30
5.4 建立DSC2基因大量表現之肺癌細胞株 30
5.5 大量表現DSC2基因會抑制肺癌細胞的遷移能力 31
5.6 大量表現DSC2基因會抑制肺癌細胞的入侵能力 31
5.7 大量表現DSC2基因會增加肺癌細胞與細胞間的黏附能力 31
5.8 大量表現DSC2會抑制肺癌細胞的增生能力 32
5.9 大量表現DSC2基因會抑制肺癌細胞群落形成的能力 32
5.10 大量表現DSC2基因會抑制肺癌細胞Anchorage-independent環境下之細胞群落形成的能力 33
5.11 DSC2基因可能影響的訊息傳遞路徑 33
5.12 建立大量表現NDRG1基因之肺癌細胞株 34
5.13 大量表現NDRG1基因會促進肺癌細胞的遷移能力 35
5.14 大量表現NDRG1會促進肺癌細胞的入侵能力 35
5.15 大量表現DSC2基因之肺癌細胞珠中增強NDRG1基因表 現 35
5.16 NDRG1基因表現會增強原本受到DSC2基因抑制肺癌細胞的遷移能力 36
5.17 NDRG1基因表現會增強原本受到DSC2基因抑制肺癌細胞的入侵能力 37
5.18在大量表現DSC2基因之肺癌細胞珠中建立穩定表現NDRG1基因之系統 37
6. 討論 38
7. 結論 43
參考文獻 71
附錄 79

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