博碩士論文 90224018 詳細資訊

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姓名 翁婉青(Wan-Ching Won)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 亞砷酸鈉誘引分裂中期停滯細胞之蛋白質體研究
(Proteomic Profiling of Arsenite-arrested Metaphase Cells)
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摘要(中) 摘 要
流行病學的研究顯示砷化物是人類致癌劑,但其致癌機轉尚待查明。在本實驗室黃錫慶博士的研究指出HeLa S3細胞經過5 μM亞砷酸鈉處理,會有大約35%的細胞停滯在有絲分裂中期,而處理staurosporine (SSP,蛋白質激酶抑制劑)之後,會促使中期停滯細胞離開有絲分裂期。本研究的目的為探討SSP如何讓亞砷酸鈉誘引的中期停滯細胞離開有絲分裂期。藉由免疫螢光染色法觀察有絲分裂期之細胞次級結構:紡錘體及DNA的構形,使用西方點墨法來偵測細胞中Pds1的含量。結果顯示亞砷酸鈉誘引的中期停滯細胞經過50 nM SSP的處理後,會促使Pds1的降解,同時也伴隨著細胞質分裂。除此之外,藉由二維電泳的分析,觀察到亞砷酸鈉誘引的中期停滯細胞,經過SSP的處理後,其蛋白質表現的改變。藉由MALDI-TOF來鑑定這些蛋白質的種類。在SSP的處理下,觀察到carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein等蛋白質的改變。另外,將nocodazole及亞砷酸鈉誘引的中期停滯細胞的紡錘體,藉由二維電泳法分析比較兩種處理下ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein等之蛋白質發生改變。這些參與有絲分裂停滯機制之蛋白質值得進一步的探討。
摘要(英) Abstract
Arsenic is a well-documented human carcinogen, primarily based on the evidence of clinical observation and epidemiological studies. Previous studies in our laboratory have shown that treatment of HeLa S3 cells with 5 μM arsenite leads to approximately 35% of the cells arrested in the mitotic metaphase. Treatment of arsenite-arrested mitotic metaphase cells with staurosporine (SSP, a protein kinase inhibitor) enforces them exiting from mitosis. In this thesis, experiments were conducted to ask how SSP enforces the mitotic exit. The microscopic structure of mitotic spindles and chromosomes were examined by immunofluorescent technique using anti-β-tubulin antibody and DAPI, and the content of Pds1 were analyzed by Western blot technique. The results show that treatment of arsenite-arrested mitotic cells with 50 nM SSP leads to degradation of Pds1 and increases in the frequency of cytokinesis. The proteins modified by SSP on arsenite arrested mitotic cells are analyzed by two-dimensional electrophoresis. These proteins are identified by MALDI-TOF technology. Several proteins are apparently modified by staurosporine treatment, for instance, carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein etc. Two-dimensional electrophoresis also detects differentially expressed proteins between the nocodazole and arsenite-arrested mitotic spindle fractions. These proteins are ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein. The involvement of these proteins in mitotic arrest warrants further investigation.
關鍵字(中) ★ 亞砷酸鈉
★ 分裂中期停滯細胞
關鍵字(英) ★ metaphase Cells
★ arsenite
論文目次 目 錄

目錄…………………………………………………………………………….…..I
『表』目錄……………………………………………………………………….IV
『圖』目錄………………………………………………………………………..V
中文摘要………………………………………………………………………....VI
英文摘要…..…………………………………………………………………….VII
第一章 緒論…..…………………………………………………………………..1
壹、砷化物的簡介…..………………………………………………….………2
一、砷的分佈與來源………………………………………………………2
二、砷化物對人類健康之影響……………………………………………2
貳、砷化物的細胞毒性與遺傳毒理………………………………………….. 3
一、砷化物的細胞毒性……………………………………………………3
二、砷化物的遺傳毒理……………………………………………………3
參、砷化物對細胞週期之影響…………………………………………………3
一、細胞週期………………………………………………………………3
二、砷化物對細胞週期之影響……………………………………………5
三、微管動力學……………………………………………………………5
四、砷化物對有絲分裂期之影響…………………………………………6
肆、Staurosporine的介紹………………………………………………………7
一、 Staurosporine的簡介…………………………………………….….7
二、 Staurosporine的作用……………………………………………..…7
伍、Pds1的介紹 ……………………………………………………………8
陸、研究動機與實驗目的………………………………………………………8
第二章 材料與方法……………………………………………………………..10
壹、 細胞培養…………………………………………………………………10
1. 細胞株…………………………………………………..……………10
2. 細胞培養………………………………………………….………….10
貳、 細胞分裂中期指數的檢測及計算…………………………..…………..10
1. 藥物處理及染色體製備……………………………………………..10
2. 細胞分裂中期指數的計算…………………………………………..11
參、 紡錘體的免疫螢光染色 …………………………………………….11
肆、 西方點墨法分析Pds1含量..……………………………………………11
1. 樣品製備………………………………………………..……………11
2. 蛋白質定量………………………………………………….……….12
3. 西方點墨法…………………………………………………………..12
伍、 二維電泳法………………..…………………………………………….12
1. 全細胞樣品製備…………………………………..…………………12
2. 紡錘體樣品製備……………………………………..………………13
3. 蛋白質裝填……………………………………..……………………13
4. 第一維等電交集電泳程式設定……………………..………………14
5. 平衡………………………………………………….……………….14
6. 第二維SDS-PAGE電泳…………………………………………….14
陸、 膠體染色法及影像處理…………………………………………………15
1. SYPRO Ruby染色法………………………………………………..15
2. 影像處理…………………………………………………………….15
柒、 膠體內分解胜肽片段……………………………………………………15
1. 挖取膠點…………………………………………………………….15
2. 膠點褪染…………………………………………………………….15
3. 膠體內分解胜肽片段……………………………………………….16
捌、 蛋白質資料庫比對………………………………………………………16
玖、 影像分析…………………………………………………………………17
第三章 結果……………………………………………………………………..18
壹、亞砷酸鈉對紡錘體型態及染色體分佈的影響………………………….18
貳、Staurosporine 對於亞砷酸鈉誘引之分裂中期停滯細胞離開有絲分裂期的影響……………………………………………………………………19
參、Staurosporine對於亞砷酸鈉誘引之分裂中期停滯細胞之紡錘體的影響…………………………………………………………………………19
肆、亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine處理後之Pds1蛋白質的表現情形……………………………………………………………20
伍、以二維電泳法觀察亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine處理後之蛋白質修飾作用………………………………………………20
陸、以二維電泳法觀察nocodazole以及亞砷酸鈉誘引之分裂中期停滯細胞內蛋白質的改變…………………………………………………………21
第四章 討論……………………………………………………………………..23
第五章 結論與展望……………………………………………………………..29
壹、結論………………………………………………………………………29
貳、展望………………………………………………………………………29
參考文獻………………………………………………………….……………...44
附錄……………………………………………………………………………... .54
『表』目錄

表1. 亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine不同時間處理後細胞
內蛋白質的變化.………………………………………………………...31
表2. 亞砷酸鈉及nocodazole誘引之分裂中期停滯細胞內蛋白質的變化…………………………………………………………………………32
『圖』目錄

圖1. 以免疫螢光染色法觀察亞砷酸鈉干擾紡錘體型態及染色體的分布
…………………………………………………………………….……..33
圖2. SSP促使亞砷酸鈉誘引之分裂中期停滯細胞離開有絲分裂期
…………………………..……………………………………………….34圖3. 以免疫螢光染色法觀察SSP促使亞砷酸鈉誘引之分裂中期停滯細胞進
行細胞質分裂…………………………………………………………...35
圖4. SSP促使亞砷酸鈉誘引之分裂中期停滯細胞進行細胞質分裂………………………………..……………………………………….36
圖5. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後Pds1蛋白之表現受抑制…………………………………………………….…………………...37
圖6. SSP處理亞砷酸鈉誘引之分裂中期停滯細胞的二維電泳膠片…..…….38
圖7. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,表現量增加的蛋白質:vinculin及glycyl-tRNA synthetase………………………………..39
圖8. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,表現量減少的蛋白質:carbamoyl-phosphate synthase……………..……….………………40
圖9. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,可能進行去磷酸化作用之蛋白質:75 kDa glucose regulated protein及heat shock 70 kDa protein 1…………………………………………………………………..41
圖10. 細胞經亞砷酸鈉及nocodazole處理後的二維電泳膠片
……………………………………………………………………………42
圖11. 細胞經亞砷酸鈉(5 µM, 24 h)及nocodazole (0.2 µM, 4 h)個別處理後,細胞間絲相關蛋白質:cytokeratin 8及vimentin的profile(側面圖)具差異性……………………………………………………………………...….43
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指導教授 李德章、黃榮南
(Te-Chang Lee、Rong-Nan Huang)
審核日期 2003-7-9
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