臺灣博碩士論文加值系統

中文摘要
三聚氰胺是一種常見的化學藥劑用於製造氨基樹脂和塑料，但目前發現除了奶粉及奶製品受三聚氰胺污染外，甚至部份日常食品、海鮮、肉類和蔬菜也已被廣泛污染三聚氰胺，還有台灣民眾外食習慣使用的美耐皿餐具遇熱也會釋出三聚氰胺。雖然成人大量飲水可把三聚氰胺隨尿液排出，但是每天低劑量重覆暴露三聚氰胺也可能對成人腎臟造成慢性傷害，然而這需要進一步的研究證明。
本研究目的在於分析三聚氰胺對人類腎臟近端腎小管細胞（HK - 2細胞）的毒性作用，我們使用WST-1 assay偵測細胞存活率，利用Hoechst 33342染色及Flow cytometry去看細胞凋亡的情形，以細胞培養液中的LDH活性去看細胞壞死，以西方墨點法觀察ERK 1/2、Bcl-2家族蛋白、Caspase 3和NF-κB等訊息傳遞路徑，用即時定量聚合酶連鎖反應觀察發炎基因IL-1β、IL-6、MCP-1及VCAM-1與生長基因TGF-β等的mRNA表現，也用CM-H2DCFDA螢光試劑去偵測細胞產生氧化壓力的情形。
研究結果指出三聚氰胺會增加ROS、促進細胞激素的表現、且會促使初期細胞凋亡因子的變化、然後誘導細胞凋亡。我們發現三聚氰胺不僅只經由尿液中晶體的形成而損害腎臟，還有可能導致細胞發炎和細胞凋亡，所以長期暴露三聚氰胺可能導致腎臟近端腎小管細胞纖維化，最終導致腎臟疾病。

Abstract
Melamine is a chemical commonly used in the manufacture amino resins and plastics. Recently, it was found in milk and milk products. Even daily foods, seafoods, meats and vegetables have been widely contaminated with melamine, as well as eating habits of people in Taiwan using melamine tableware from which melamine case of heat will be released. While the adults drink lots of water can be excreted in the urine of melamine, but repeated daily exposure to low doses of melamine may cause chronic kidney damage in adults; however, it needs further studies to prove.
The purpose of this study is to analyze the toxicity of melamine on human renal proximal tubular cells (HK - 2 cells). We used WST-1 assay to detect cell viability, used Hoechst 33342 stain and flow cytometry to see cell apoptosis, used measurement of LDH activity to detect cell necrosis, and used Western blotting to observe the protein expression of ERK 1/2, Bcl-2 family proteins, Caspase-3 and NF-κB. Real-time polymerase chain reaction was used to detect the mRNA expression of inflammatory genes including TGF-β, IL-1β, IL-6, MCP-1 and VCAM-1. The CM-H2DCFDA fluorescent reagent was used to detect ROS generation from the cells.
Our study demonstrated that melamine can stimulate ROS generation, increase the expression of inflammatory factors, regulate transcriptional factors and apoptotic related factors, and induce HK-2 cell apoptosis. We found that melamine could cause kidney damage not just by the formation of crystals; melamine may also lead to inflammation and cell apoptosis. In conclusion, the long-term exposure to melamine can cause kidney proximal tubular cells injury and eventually lead to kidney disease.

目 錄

中文摘要………………………………………………………………….3
英文摘要………………………………………………………………….4
壹 緒論…………………………………………………………………...5
一、三聚氰胺毒性作用引發腎衰竭………………………………….6
二、三聚氰胺與日常食物、食物器皿關係…………………………12
三、腎臟細胞傷害之病理機轉與發炎反應…………………………13
四、研究目的…………………………………………………………20
貳 材料與方法…………………………………………………………..22
一、細胞培養…………………………………………………………23
二、細胞存活率測定…………………………………………………24
三、Hoechst 33342螢光染色………………………………………...25
四、流體細胞免疫分析………………………………………………26
五、ROS之測定………………………………………………….......27
六、細胞RNA之萃取……………………………………………….28
七、即時定量聚合酶連鎖反應……………………………………....30
八、細胞蛋白質之萃取……………………………………………....31
九、西方墨點法………………………………………………………32
參 研究結果……………………………………………………………..36
肆 討論與結論…………………………………………………………..43
伍 附圖…………………………………………………………………..48
陸 參考文獻……………………………………………………………..66

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