- 學位論文
探討橘黴素對人類腎臟細胞的癌化能力
Evaluation of Carcinogenic Effects of Citrinin on Human Kidney Cells
摘要
橘黴素(Citrinin, CTN)是一種黴菌的二級代謝物，普遍存在於被污染的穀類製品中，CTN已被證實會對模式動物產生腎毒性及肝毒性，且會造成急性腎管細胞壞死及腎臟腫大的現象。在本篇研究中我們利用人類胚胎腎臟細胞(HEK293)及人類腎臟近曲小管表皮細胞(HRPTEC) 進行CTN毒理機轉的研究。我們將HRPTEC初代細胞進行不死化(immortalization)後以不同濃度的CTN處理，結果發現CTN會使染色體數目異常的細胞比例增加，也會誘發基質金屬蛋白酶第九型(matrix metalloproteinases, MMP 9) 的活性。另一方面，經CTN處理過後的HEK293細胞能夠在軟瓊脂膠上形成細胞聚落，表示CTN會使腎臟細胞具有非貼附性生長的能力(anchorage-independent growth)；依CTN處理時間的長短將從軟瓊脂膠上分離出來的細胞株分別建立為CTN短期處理細胞株及CTN長期處理細胞株，不論是短期或是長期處理的細胞株都具有遷移及侵入的能力。此外，利用MTT assay及BrdU細胞增生試驗也發現CTN短期處理細胞株有細胞增生的現象。CTN的處理導致細胞株形態變得較為細長，也會降低CTN短期/長期處理細胞株中表皮細胞指標E-cadherin的蛋白質表現量，並且會增加間質細胞指標Snail、Slug的mRNA表現量及Vimentin的mRNA與蛋白質表現量。綜合以上結果可知CTN可能會使人類腎臟細胞株產生癌化及epithelial-mesenchymal transitions(EMT)的現象。
並列摘要
Mycotoxin Citrinin (CTN) is a secondary metabolite of fungi, also a frequent natural contaminant of cereals. CTN acts as a nephortoxin or hepatotoxin and causes acute tubular necrosis and kidney enlargement in animal models. In the present study, the toxicological mechanism of CTN was investigated by applying human embryonic kidney 293 (HEK293) cells and human renal proximal tubular epithelial cells (HRPTEC) as models. Treatment of immortalized HRPTEC cells with CTN increased the ratio of abnormal chromosome numbers and MMP 9 activity. On the other hand, the exposure of HEK293 cells to CTN induced cell colony formation on soft agar, suggesting that CTN can cause anchorage independent growth of kidney cells. Depending on the CTN exposured time, the CTN-treated HEK293 cells, isolated form soft agar, were grouped into long-term and short-term cell lines. Both the long-term and short-term cell lines had gained the migration and invasion abilities. In addition, the proliferation of short-term cell lines was increased in MTT and BrdU cell proliferation assays. HEK293 cells after CTN treatment also changed its cell morphology into spindle-like phenotype. CTN not only decreased the protein level of E-cadherin, an epithelial marker, in long-term/short-term cell lines, but also increased the mRNA or protein levels of mesenchymal markers, including snail, slug and vimentin. Taken together, the data suggested that CTN may cause carcinogenicity and epithelial-mesenchymal transitions(EMT) in human kidney cells.