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研究生(外文):Sing Ru Lin
論文名稱(外文):G6PD-status affects TNF-α-induced signal transduction in lung carcinoma (A549) cells
指導教授(外文):P. T. Y. ChiuC. M. Yang
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葡萄糖六磷酸去氫酶(Glucose-6-phosphate dehydrogenase, G6PD)最主要的功能就是產生NADPH,進而維持細胞內氧化還原的平衡。當細胞遭受病原體感染時,會釋放出TNF-α等細胞激素,而TNF-α所誘導的訊息傳遞路徑會透過NF-κB及MAPK pathway來產生發炎相關蛋白質COX-2,進而調控細胞功能。目前TNF-α所誘導訊息傳遞路徑已建立,然而G6PD缺乏所造成氧化還原不平衡是如何影響TNF-α所誘導之訊息傳遞並不清楚,故本實驗室以G6PD knockdown的A549細胞株,作為氧化還原狀態不平衡的細胞模式,並進一步探討G6PD缺乏的細胞對TNF-α誘導之訊息傳遞路徑所造成的影響。本實驗室目前研究發現G6PD-knockdown的A549細胞與對照組相比,因缺乏NADPH,在TNF-α處理後,經由NADPH oxidase所產生的superoxide亦低於對照組。進一步追蹤下游MAPK訊息路徑,發現G6PD-knockdown A549其phospho-p38, ERK, JNK磷酸化的量都明顯減少,而發炎相關蛋白質COX-2的表現時間有明顯延遲的現象。COX-2蛋白質延遲及p38磷酸化減少的現象皆在處理apocynin的對照組細胞中亦可以觀察到相似的結果。故本實驗室推測TNF-α誘發的訊息傳遞路徑,例如MAPK及COX-2的表現,會受細胞內還原/氧化狀況所影響。
Glucose-6-phosphate dehydrogenase (G6PD) , the key regulatory enzyme in the pentose phosphate pathway, provides reducing power to all cells in the form of NADPH to meet the cellular needs for reductive biosynthesis and maintenance of the cellular redox status. We have previously reported that G6PD-deficient cells suffer high oxidative stress and more recently, we have found that these cells are more susceptible to viral infection than normal counter parts. However, how G6PD deficiency may affect cellualr signaling remains elusive. Toward this end, we used G6PD-knockdown lung carcinoma cells (A549) as cell model to investigate the effects of cellular redox status on TNF-α signaling pathway. We found that G6PD-knockdown cells produced less superoxide upon 15 ng/ml TNF-α treatment at early stage than that produced by normal cells. In addition, G6PD-knockdown A549 cells showed less phosphorylation of p38, ERK and JNK proteins comparing to control cells. By using western blotting technique, we observed a delayed pattern of inflammatory protein Cox-2 expression upon TNF-α treatment at different time course in G6PD-knockdown A549 cells comparing to control cells. Furthermore, similar delayed Cox-2 expression pattern in control cells was observed by apocynin pretreatment. Taken together, these data support the notion that cellular redox status in host cells is an important factor in TNF-α signal transduction, and may have implications in tumor biology and cancer therapy.

誌謝 ------ iv
中文摘要------ v
英文摘要 ------ vi
目錄 ------ vii
圖表目錄 ------ viii
前言 ------ 1
實驗設計 ------ 6
實驗材料 ------ 7
實驗方法------ 9
實驗結果 ------ 14
討論 ------ 20
參考文獻------ 29


圖一、G6PD-knockdown A549 cell 其G6PD活性及蛋白質表現量與NADPH含量皆顯著比對照組A549來得少------------------------------36
圖二、G6PD-knockdown A549 cell 與對照組A549的TNFR1的表現量並無明顯差異------------------------------------------------38
圖四 、 G6PD活性不同的A549細胞在TNF-α處理不同時間點後,p-P38, ERK, JNK的磷酸化變化-------------------------------------40
圖六、G6PD活性不同的A549細胞在TNF-α處理不同時間點後,加入p-P38 inhibitor(SB2030580)後,COX-2表現量變化-------------------43
圖七、G6PD活性不同的A549細胞在TNF-α處理不同時間點後,加入MEK1/2 inhibitor(PD98059)後,COX-2表現量變化---------------------44
圖九、對照組A549細胞處理apocynin與TNF-α後,p38 MAPK的磷酸化變化-----------------------------------------------------------46
圖十一、G6PD-knockdown細胞中TNF-α 所誘導的訊息傳遞路徑--------------------------------------------------------------------48
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