VHL knockdown HK-2 cells induce macrophage endothelial extravasation

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题名:	VHL knockdown HK-2 cells induce macrophage endothelial extravasation
作者:	劉耕維;Liu, Keng-Wei
贡献者:	系統生物與生物資訊研究所
关键词:	巨噬細胞;VHL;macrophage extravasation
日期:	2018-01-22
上传时间:	2018-04-13 11:02:12 (UTC+8)
出版者:	國立中央大學
摘要:	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。;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.
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