臺灣博碩士論文加值系統

轉錄作用中，tRNA負責攜帶特定的胺基酸至mRNA上與其密碼子相互配對，進行蛋白質胜鏈的合成。在tRNA的作用機制上，研究指出tRNA上wobble反密碼子之鹼基修飾與mRNA密碼子辨識功能有關，其中第34號wobble位置擁有最多種類之鹼基修飾。鹼基修飾之一是wobble位置的硫醇鹼基修飾，其硫原子的來源與利用與硫鐵蛋白有關，後者之含量則與鐵營養狀況有關。本研究將深入了解鐵營養對tRNA分子形式與功能的影響，首先進行營養depletion-repletion實驗，以tRNALys與tRNAGlu為目標，檢視鐵營養對肌肉組織tRNA硫醇鹼基修飾的劑量效應。針對不同含鐵濃度去偵測大鼠肌肉及肝臟tRNA不同劑量對於tRNALys與tRNAGlu探針的表現，接續欲探討不同濃度硫酸亞鐵之補充（6、12、18、24、35 ppm）對於膳食缺鐵大鼠tRNA表現量及硫醇修飾化程度之關係。實驗步驟分為t RNA定性與定量，以及動物實驗兩大項目。實驗結果顯示，北方點墨分析法及APM-北方點墨分析法分別為偵測肌肉及肝臟組織tRNA表現量及tRNA硫醇鹼基修飾作用之重要工具，不論是在肌肉及肝臟組織， tRNAGlu的探針訊號遠高於tRNALys。另外，本研究證實膳食鐵劑之補充會增加肌肉細胞質tRNALys及tRNAGlu表現量及其硫醇鹼基修飾化作用，但對於肝臟組織，膳食鐵劑量補充對於提高肝臟細胞質tRNAGlu表現量及其硫醇鹼基修飾較為明顯，另外，本實驗也確認了膳食鐵劑對於肌肉含硫醇鹼基修飾tRNA分子之表現為專一性反應。

In the process of transcription, transfer RNAs (tRNAs) take responsibility in bringing specific amino acid to its corresponding codon on the messenger RNA (mRNA) and the formation of protein peptides. With the advance of research, in addition to its role in transcription, more diverse active molecular and biological functions of tRNAs have been unveiled. Recent studies have demonstrated that tRNAs have the ability to regulate proliferation and apoptosis and are also implicated in some diseases. Some studies indicated that modified anticodons, on wobble position 34 in particular, play a crucial part in recognizing mRNA. One important tRNA post-transcriptional modification is thio-modification, in which the source and utilization of sulfur are related to the iron-sulfur protein in dietary iron intake. Therefore, in this study, we investigate the effects of iron repletion on tRNA molecular type and function. In the beginning, we used iron depletion-repletion by tRNALys and tRNAGlu as probes to survey dose response of tRNA thio-modification in muscle and liver tissues. We measured the expression of tRNALys and tRNAGlu under different dosage of iron in rat muscle and liver and examined the correlation between tRNA expression and thio-modification with various concentration of FeSO4 repletion after iron deficiency. Two major experimental methods included tRNA qualitative and quantitative analyses and animal model study. We show in this study that Northern blotting and APM Northern blotting are vital tools for the detection of tRNA expression and thio-modification in rat muscle and liver tissues. It is interesting to note that the probe’s signal of tRNAGlu is much stronger than that of tRNALys no matter in muscle or liver tissue. Moreover, repletion of dietary iron rescues both the tRNALys and tRNAGlu expression and thio-modification in rat muscle whereas in rat liver it selectively increases the tRNAGlu expression and thio-modification. Lastly, this study confirmed that dietary iron affects specifically the thio-modified tRNA expression in rat muscle.

中文摘要 i
Abstract ii
縮寫表 iv
第一章 文獻回顧 1
一、 tRNA之簡介 1
(一) tRNA之結構與種類 1
(二) tRNA之生合成機制 2
(三) tRNA之功能及基因表現調控 3
二、 tRNA轉錄後修飾之重要性 4
(一) tRNA分子之鹼基修飾 4
(二) tRNA硫醇鹼基修飾作用及影響 5
(三) tRNA硫醇鹼基修飾之機制 6
三、 硫鐵蛋白與tRNA硫醇鹼基修飾之相關性 7
(一) 硫鐵蛋白之種類及生理功能 7
(二) 硫鐵蛋白之生合成系統及調節機制 8
(三) 真核生物ISC系統對鐵恆定之影響 12
(四) ISC及CIA系統對於tRNA硫修飾作用扮演之角色 13
(五) 硫鐵蛋白生合成異常與疾病之相關性 15
四、 膳食缺鐵對生物體之影響 16
五、 研究動機與目的 16
第二章 材料與方法 18
一、 實驗設計 18
二、 tRNA定性與定量條件之建立 18
(一) 肌肉組織小片段RNA分子之萃取與分離 18
(二) 肌肉組織tRNA表現量之分析 20
(三) 肌肉組織tRNA硫醇鹼基修飾作用分析 22
(四) 統計分析 22
三、 動物實驗 23
(一) 動物飼養 23
(二) 飼料配製 23
(三) 尾巴採血與犧牲收養 24
(四) 血液鐵指標之測定 24
(五) 肌肉組織之採集 26
(六) 統計分析 26
第三章 結果 30
一、 動物實驗 30
(一) 缺鐵期及再生期大鼠之生長狀況 30
(二) Wister雄性大鼠之鐵營養指標 30
(三) 大鼠膳食缺鐵之臨床表徵及生理影響 31
二、 建立tRNA定性與定量之各項條件 31
(一) Wister雄性大鼠肌肉及肝臟小片段RNA電泳分析 31
(二) 確立小片段RNA於濕式轉印槽之條件 31
(三) 不同劑量對大鼠肌肉及肝臟小片段RNA於tRNALys與tRNAGlu之影響 32
(四) Stripping次數對tRNA表現量之影響 32
(五) 大鼠肌肉細胞質tRNA表現之測試 33
(六) APM對於大鼠肌肉含硫醇鹼基tRNA之硫醇修飾作用之測定 33
三、 膳食補鐵營養狀況對於大鼠肌肉及肝臟tRNA表現及硫醇鹼基修飾作 用之影響 34
(一) 肌肉細胞質中tRNA之表現 34
(二) 肌肉細胞質中硫醇鹼基修飾作用之表現 34
(三) 肝臟細胞質中tRNA之表現 35
(四) 肝臟細胞質中硫醇鹼基修飾作用之表現 35
四、 膳食補鐵營養況狀對於大鼠肌肉tRNA表現之專一性反應 36
第四章 討論 63
一、 北方點墨分析法及APM-北方點墨分析法分別為組織tRNA表現量及tRNA硫醇鹼基修飾作用之重要工具 63
二、 補充膳食鐵對於大鼠肌肉及肝臟細胞質tRNA表現量及tRNA硫醇鹼基修飾作用之影響 63
三、 膳食缺鐵對於生物體tRNA表現及硫醇簡基修飾作用之調控機制 64
四、 膳食鐵對於肌肉細胞質含硫醇鹼基修飾之tRNA表現為專一性反應 65
五、 tRNA於氧化壓力之調控作用 66
六、 結論 67
第五章 參考文獻 68
附錄 83

徐韻欣（2010）膳食鐵營養狀況對於大鼠肌肉硫醇鹼基修飾tRNA的表現 與其修飾作用之影響. 輔仁大學營養科學系碩士論文

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