VHL knockdown HK-2 cells induce macrophage endothelial extravasation

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劉耕維(Keng-Wei Liu) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

(VHL knockdown HK-2 cells induce macrophage endothelial extravasation)

相關論文

★ 由基因微陣列分析發炎與腎臟細胞癌發生之機制	★ VHL基因突變在癌前期的組織發炎機制
★ VHL剔除模型之轉錄體差異以及台灣透明細胞腎細胞癌族群之特定基因體變異之研究	★ ITPR2, an ER calcium channel, regulates ER stress and inflammatory response in pre-cancerous kidney tubule cells
★ 透明腎臟細胞癌發生前期與組織發炎之關係研究	★ VHL與KIM-1的功能關係研究
★ 血管內皮細胞在腫瘤微環境中促進透明腎細胞癌形成之研究	★ Study of the Interaction between VHL/Vhlh Deficient Kidney Epithelial Cells and Macrophages—Relevance to the Development of Clear-Cell Renal Cell Carcinoma
★ 應用大腸桿菌與酵母菌蛋白質體晶片系統性分析抗菌肽及抗生素作用之目標蛋白質	★ Analysis of Gene Expression of Chronic Obstructive Pulmonary Disease and Chronic Kidney Disease to Illuminate Chronic Inflammation Associated with Tumor Microenvironment and Potential Treatment

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摘要(中)

腎細胞癌對大多數化學治療或是放射治療都具有抗性，逢希伯-林道(Von Hippel-Lindau, VHL)是著名的透明細胞腎細胞癌(clear-cell renal cell carcinoma, ccRCC )的腫瘤抑制基因。近來發現，慢性發炎會導致透明細胞腎細胞癌。而在我們的VHL基因剔除小鼠模式中也展示了喪失VHL蛋白質的功能將導致嚴重的發炎與纖維化現象。在這次的研究中，我們在transwell測定中共同培養VHL基因下調或正常的人類非癌症腎臟細胞(human non-cancerous kidney cell line, HK-2)、人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial cells, HUVECs)以及使用單核細胞(monocyte, THP-1)經丙二醇甲醚醋酸酯(phorbol myristate acetate, PMA)引發而成的巨噬細胞(macrophage)等三種細胞來模擬巨噬細胞的內皮細胞外滲現象。在模擬現象的實驗中，單層的人類臍靜脈內皮細胞被培養在多孔膜上，在形成單層膜後加入巨噬細胞。而實驗的結果表示，在VHL基因下調的人類腎臟細胞組別中有較多的巨噬細胞穿越過人類臍靜脈細胞。而後我們更進一步地使用Human Cytokine Array找出五種可能具有引起巨噬細胞的內皮細胞外滲的發炎相關細胞因子。

摘要(英)

Renal cell carcinoma is resistant to most chemotherapy and radiation therapy. Von Hippel-Lindau (VHL) is a well-known tumor suppressor gene of clear-cell renal cell carcinoma (ccRCC). Recent findings have indicated that ccRCC is caused by chronic inflammation. Our VHL gene knock out mouse model also demonstrates that the immediate consequence of VHL loss-of-function is severe inflammation and fibrosis. In this research, we mime a situation of macrophage endothelial extravasation by using transwell assay and co-culture of 3 kinds of cell lines: human non-cancerous kidney cell line HK-2 (with or without VHL knockdown), Human Umbilical Vein Endothelial Cells (HUVECs), and macrophage that is induced from monocyte precursor THP-1 by phorbol myristate acetate (PMA). In this assay, a monolayer of HUVECs is grown on the porous membrane, on which differentiated THP-1 is then seeded. The capability of kidney tubule cells to attract THP-1 cells to pass through the HUVEC monolayer (endothelial extravasation) is measured, comparing VHL+ and VHL knockdown. We found that VHL knockdown HK-2 cells could better induce macrophage endothelial extravasation. Further, we used Human Cytokine Array to identify five inflammation-related cytokines that could recruit macrophages.

關鍵字(中)

★ 巨噬細胞

關鍵字(英)

★ VHL
★ macrophage extravasation

論文目次

摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖目錄 List of Figures v
表目錄 List of Tables vi
Chapter 1 Introduction 1
1-1 Von Hippel-Lindau Syndrome 1
1-2 Renal Cell Carcinoma 4
1-3 VHL Mutant Leads to Precancerous Tissue Inflammation in ccRCC 6
1-4 Macrophages Polarization and Tumor-Associated Macrophage (TAM) 9
Chapter 2 Materials and Methods 12
2-1 Reagents 12
2-1-1 12-O-Tetradecanoylphorbol-13-acetate 12
2-1-2 Other Reagents 13
2-1-3 Reagents for Cell Culture 14
2-2 Methods 15
2-2-1 DNA Extraction 15
2-2-2 Transfection 16
2-2-3 Transwell Assay 17
2-2-4 Protein Isolation 18
2-2-5 Western Blot Analysis 19
2-2-6 Crystal Violet Staining of Transwell 20
2-2-7 Human Cytokine Array 21
Chapter 3 Results 23
Chapter 4 Conclusion and Discussion 34
Chapter 5 Reference 37

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指導教授

徐沺(Tien-Hsu)

審核日期

2018-1-22

推文