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作者(中文):陳麗芬
作者(外文):Chen, Li-Fen
論文名稱(中文):鎘對腎臟細胞中金屬硫蛋白、穀胱甘肽與硫氧化還原蛋白-1表現之相關性的研究
論文名稱(外文):Studies on the Relationship among Metallothionein, Glutathione and Thioredoxin-1 Expressions in HEK293 Cells under Cadmium Stimulation
指導教授(中文):林立元
指導教授(外文):Lin, Lih-Yuan
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學號:9580531
出版年(民國):98
畢業學年度:97
語文別:中文
論文頁數:53
中文關鍵詞:
外文關鍵詞:MTGSHTRX 1
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鎘是環境中常見的污染物質,透過ROS的產生使細胞遭受氧化逆境進而導致細胞死亡。Metallothionein (MT)與glutathione (GSH) 是解除細胞內鎘毒性的主要生物分子。鎘會與thioredoxin (Trx)的活性中心結合,抑制Trx活性進而影響細胞死亡路徑。為了要探討鎘如何影響MT基因、GSH與Trx三者之間的相互關係,我們將HEK293細胞處理鎘,發現γ-glutamylcysteine synthetase (γ-GCS )表現量降低並且伴隨細胞內GSH消耗。鎘誘導MT基因大量表現,造成Trx 1蛋白質表現量減少,並且促使Trx 1氧化。接著我們利用N-acetylcysteine (NAC)與DL-buthionine-[S,R]-sulfoximine (BSO)來改變細胞內GSH含量,並在鎘的共同處理下觀察MT基因與Trx 1的變化。由實驗結果發現NAC可以恢復鎘處理造成的GSH消耗,降低MT基因的誘導與Trx 1的氧化,BSO則進一步導致Trx 1完全氧化。隨後將質體送入HEK293細胞中表現Trx 1或利用RNAi技術knockdown Trx 1表現,探討MT基因與GSH的相互關係。細胞內GSH含量不受Trx 1蛋白質表現而影響,驗證GSH與Trx是獨立的兩個系統。Knockdown Trx 1並與鎘共同處理之下,發現MT基因受到誘導的程度降低,可能是間接影響其他蛋白質活性,進而改變MTF-1調控MT基因的表現,其明確機制需更多的研究來加以證實。
中文摘要 I
Abstract II
謝誌 III
目次 IV
圖次 V
緒論 1
實驗材料與方法 9
1. 化學藥品及抗體 9
2. 細胞株培養 9
3. 細胞質RNA萃取 (Extraction Cytoplasmic RNA) 10
4. 甲醛變性瓊脂凝膠電泳 (Formaldehyde/Denaturating Agarose Gel Electrophoresis) 11
5. 反轉錄 (Reverse Transcription) 11
6. 即時定量聚合酶鏈鎖反應 (Quantitative Real-time PCR) 12
7. Quantitative Real-time PCR引子效率測定及相對定量的數學運算 12
8. 細胞轉型 (Transfection) 13
9. 蛋白質定量 13
10. 西方墨點法 14
11. Redox Western Blot Analysis 15
12. 細胞中Glutathione (GSH)含量分析 16
13. RNA Interference 17
14. MTT試驗 17
結果 18
1. 不同濃度鎘處理對HEK293細胞內GSH含量的影響 18
2. 不同濃度鎘對HEK293細胞中γ-GCSh蛋白質表現量的影響 18
3. 不同濃度鎘對HEK293細胞中MT基因表現量的影響 19
4. 不同濃度鎘對HEK293細胞中Trx 1蛋白質表現量影響 20
5. 不同濃度鎘處理HEK293細胞對Trx 1氧化還原狀態的影響 20
6. 改變細胞中GSH含量進而觀察mt與Trx 1之間的變化 21
7. 改變細胞中Trx 1表現量來觀察對GSH的影響 23
8. 改變細胞中Trx 1表現量來觀察對mt的影響 24
9. Trx 1 siRNA與鎘對HEK293細胞存活性的影響 25
討論 26
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