博碩士論文 90224016 詳細資訊




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姓名 張瑞娟(Jui-Chuan Chang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 低劑量亞砷酸鈉誘引第一型血紅素氧化酶調控路徑之研究
(Signaling pathways involved in Heme Oxygenase-1 induction by low doses of arsenite)
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摘要(中) 中文摘要
砷化物是人類的致癌物質,會引起如動脈粥狀硬化、烏腳病等一些心臟血管疾病。第一型血紅素氧化酶( Heme oxygenase – 1, HO-1) 能被許多物質所誘導出來,包括血色素、氧化性壓力、熱刺激、生長因子以及一些重金屬,如砷化物,鎘金屬。其功能是代謝血紅素產生鐵離子、膽紅素、與一氧化碳。然而其中膽紅素具有抗氧化的功能,因此第一型血紅素氧化酶的生成,被認為是當細胞遭受到氧化性傷害時,被誘導出來保護細胞免於氧化性傷害。研究發現在牛的大動脈血管細胞與人類成纖細胞中,低濃度(1 ?M)亞砷酸鈉會誘引第一型血紅素氧化酶蛋白質的表現。為了探討其誘導途徑與參與的分子,本文採用了許多抑制劑。其中轉錄型抑制劑,adriamycin 與actinomycin D能明顯的抑制低濃度亞砷酸鈉誘引第一型血紅素氧化酶蛋白質的表現,因此低濃度的亞砷酸鈉誘導第一型血紅素氧化酶,是先經由轉錄,再經由轉譯成蛋白質之路徑。其他抑制劑如Calphostin C (蛋白質激酶 C,PKC抑制劑); H8 和H89(蛋白質激 A ,PKA抑制劑); quinacrine(磷質脂A2 抑制劑); BATPA/AM(鈣離子螯合劑),皆能個別的抑制低濃度亞砷酸鈉誘引第一型血紅素氧化酶蛋白質的表現。然而其中Quinacrine與BATPA/AM的抑制能力似乎是抑制RNA的形成。因此quinacrine 和BATPA/AM抑制亞砷酸鈉誘導第一型血紅素氧化酶的能力,與抑制磷質脂A2和鈣離子的訊息傳遞之路徑無關。而在PKC抑制劑方面,另外使用Bisindolylmaleimide I、D-erythio-sphingosine、Chelerythrine chloride卻不能有效抑制被誘引第一型血紅素氧化酶蛋白質表現,因此Calphostin C似乎也並非單純經由抑制PKC的路徑去抑制低濃度亞砷酸鈉誘引第一型血紅素氧化酶的表現。目前的結果指出,在內皮細胞與人類成纖細胞中,PKA 似乎參與低濃度亞砷酸鈉誘引第一型血紅素氧化酶蛋白質調控路中。然而還是需要再做進一步的證實。
摘要(英) Abstract
Arsenite (As (III)) is a human carcinogen, also known to increase the risk of cardiovascular diseases, such as atherosclerosis and blackfoot disease. Heme oxygenase-1 (HO-1) is induced by a variety of agents, such as heme, oxidative stress, heat shock, cytokines, and some heavy metals (arsenite and cadmium). HO-1 catalyzes heme degradation into iron, biliverdin, and carbon monoxide. Biliverdin is subsequently converted to bilirubin by bilirubin reductase since bilirubin has antioxidant properties, HO-1 is considered to play a protective role against oxidative injury. In this thesis, how HO-1 is induced by low dose of sodium arsenite (< 1 ?M) in bovine aortic endothelial cells and human fibroblasts is investigated. To examine which pathway or signaling molecule involved in low dose sodium arsenite-mediated induction of HO-1, several inhibitors we were used adopted in this study. The results show that both adriamycin and actionmycin D decrease 1 ?M sodium arsenite-induced HO-1 expression, indicating that HO-1 expression induced by 1 ?M sodium arsenite is via transcription and translation. Pre-treatments of BAEC and HFW with Calphostin C (an inhibitor of protein kinase C), H8 and H89 (inhibitor of protein kinase A), quinacrine
(an inhibitor of phospholipase A2), BATPA/AM (an calcium chelater) reduce HO-1 expression levels by 1 ?M As (III). Unfortunately, quinacrine and BATPA/AM also inhibit RNA synthesis. The inhibitory effects of quinacrine and BATPA/AM on arsenite-induced HO-1 expression are unlikely due to inhibition of phospholipase A2 and calcium, respectively. Since other inhibitors of PKC family, such as bisindolylmaleimide I, D-erythio-sphingosine and chelerythrine chloride showed no effect on HO-1 induction by 1 ?M As (III), therefore Calphostin C is also not simply via PKC inhibition to decrease HO-1 expression. The present results imply that PKA is probably involved in low dose sodium arsenite-induced HO-1 expression in BAEC and HFW. However, further confirmation is required.
關鍵字(中) ★ 第一型血紅素氧化酶
★ 亞砷酸鈉
關鍵字(英) ★ arsenite
★ Heme Oxygenase-1
論文目次 目錄

目錄……………………………………………………………………………………I
『圖』目錄……………………………………………………………………………IV
『表』目錄…………………………………………………………………………..VI
專有名詞對照表……………………………………………………………………VII
中文摘要………………………..…………………………………………………VIII
英文摘要………………………………………………………..……………………X
第一章 緒論
Ⅰ、砷化物…………………………………………………………………………. 1
壹、砷化物的來源和分佈………………………………………………………..1
貳、砷化物的種類與攝入代謝…………………………………………………..2
參、砷化物的毒性………………………………………………………………..2
肆、砷化物與周狀動脈硬化之關係……………………………………………5
Ⅱ、血紅素氧化酶Heme oxygenase (HO)………………………………………….6
壹、血紅素氧化酶的機制………………………………………………………..6
貳、血紅素氧化酶的異構酶與組織特異性……………………………………..7
參、血紅素氧化酶的生化和生理功能……………………..……………………7
Ⅲ 、研究動機與實驗目的…………………………………………………………8
第二章 材料與方法
Ⅰ、實驗材料………………………………………………………………………10
壹、細胞株………………………………………………...…………………….10
貳、antibody………………………………………………….…………………10
參、抑制劑………………………………………………………………………10
Ⅱ、實驗方法………………………………………………………………………11
壹、細胞株的培養……….……………………………………...........................11
貳、藥物處理處理.………………………………………………………..…...12
參、細胞收集…………….……………………………………………………...12
肆、蛋白質定量………………………………………………………………....12
伍、十二硫酸脂鈉-多聚丙烯醯胺膠體電泳分析(sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE).......................................13
陸、西方點墨法(Western boltting)…………………………..............................13
柒、 RNA合成的測定(uridine incorporation)………….……………...……..13
第三章 結果
壹、亞砷酸鈉誘導第一型血紅素氧化酶蛋白質的表現……………………...14
貳、轉錄型抑制劑(transcriptional inhibitor)對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響…………………………………………..14
參、Staurosporine對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………………………………..15
肆、蛋白質激酶C (protein kinase C)抑制劑對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響…………………………………………..15
伍、蛋白質激酶A (protein kinase A)抑制劑對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響…………………………………………...17
陸、酪胺酸激酶(tyrosine kinase)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………..17
柒、p38抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響……………………………………………………………..18
捌、ERK抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響……………………………………………………………..18
玖、PI-3 kinase抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………………..…………18
拾、PLA2抑制劑對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響……………………………………………………….……………….19
拾壹、5′脂氧化酶(5’-lipoxygenase )與12′脂氧化酶(12’- lipoxygenase)抑制劑對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響……..19
拾貳、測定PLA2抑制劑Quinacrine對細胞嵌入尿嘧啶之放射線標定的影響([H]3 – Uridine incorporation)……………………………………………..20
拾參、細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對低濃度亞砷酸鈉誘導第一型血紅素氧化酶蛋白質表現之影響…………………………..21
拾肆、測定細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對細胞嵌入尿嘧啶之放射線標定的影響………………………………………………..22
第四章 討論…………………………………………………………………………24
第五章 結論…………………………………………………………………………30 參考文獻…………..………………………………………………………………....54
『圖』目錄

圖一、
第一型血紅素氧化酶的代謝途徑與代謝產物在內皮細胞與肌肉平滑肌細胞之關係圖…………………………………………...
31
圖二、 亞砷酸鈉誘導BAEC與HFW細胞第一型血紅素氧化酶蛋白質的表現……………………………………………………………...
32
圖三、 轉錄型抑制劑(transcriptional inhibitor)對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素養化酶蛋白現之影響………………….
33
圖四、 廣效型抑制劑(Staurosporine)對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………….......
34
圖五、 蛋白質激酶C (protein kinase C)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………….
35
圖六、 蛋白質激酶C (protein kinase C)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………….
36
圖七、 蛋白質激酶C (protein kinase C)抑制劑對低濃度亞砷酸鈉誘導BAEC細胞第一型血紅素氧化酶蛋白質表現之影響……………
37
圖八、 蛋白質激酶C(protein kinase C)抑制劑對低濃度亞砷酸鈉誘導BAEC細胞第一型血紅素氧化酶蛋白質表現之影響……………
38
圖九、 蛋白質激酶A (protein kinase A)抑制劑對低濃度亞砷酸鈉誘導BAEC細胞第一型血紅素氧化酶蛋白質表現之影響……………
39
圖十、 蛋白質激酶A (protein kinase A)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………….
40
圖十一、 酪胺酸激酶 (tyrosine kinase)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………….
41
圖十二、 p38抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………………...
42
圖十三、 ERK抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………………...
43
圖十四、 PI-3 kinase抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………...
44
圖十五、 轉錄型抑制劑(transcriptional inhibitor)對低濃度亞砷酸鈉誘BAEC與HFW細胞第一型血紅素氧化酶蛋白質表現之影響…..
45
圖十六、 PLA2抑制劑與其下游5′脂氧化酶(5’-lipoxygenase )、12′脂氧化酶(12’- lipoxygenase)抑制劑對低濃度亞砷酸鈉誘導BAEC細胞第一型血紅素氧化酶蛋白質表現之影響………………………
46
圖十七、 PLA2抑制劑與其下游5′脂氧化酶(5’-lipoxygenase )、12′脂氧化酶(12’- lipoxygenase)抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響………………………...
47
圖十八、 測定Quinacrine對細胞嵌入尿嘧啶之放射線標定的影響……… 48
圖十九、 測定細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對低濃度亞砷酸鈉誘導HFW細胞第一型血紅素氧化酶蛋白質表現之影響……………………………………………………………...
49
圖二十、 測定細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對低濃度亞砷酸鈉誘導BAEC細胞第一型血紅素氧化酶蛋白質表現之影響…………………………………………………………...
50
圖二十一、 測定細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對HFW細胞嵌入尿嘧啶之放射線標定的影響……………………
51
圖二十二、 測定細胞內外鈣離子螯合劑與抑制鈣離子傳輸之抑制劑對BAEC細胞嵌入尿嘧啶之放射線標定的影響…………………...
52
『表』目錄
表一、各種抑制劑對於亞砷酸鈉誘引第一型血紅素氧化酶之影響…………….53
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指導教授 黃榮南、李德章
(Rong-Nan Huang、Te-Chang Lee)
審核日期 2003-7-11
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