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博碩士論文 etd-0113111-173303 詳細資訊
Title page for etd-0113111-173303
論文名稱
Title
人體C1酯酶抑制蛋白對乳癌細胞增生與遷移的影響
Enhancement of growth and migration of human breast cancer cell (MDA-435S) by human C1 inhibitor
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
84
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2010-07-16
繳交日期
Date of Submission
2011-01-13
關鍵字
Keywords
增生與遷移、人體C1酯酶抑制蛋白、乳癌細胞
breast cancer cell, growth and migration, human C1 inhibitor
統計
Statistics
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中文摘要
人類第一補體抑制劑(C1 inhibitor, C1-inh) 可以抑制第一補體蛋白次單元C1s及C1r的活性而達到調節古典路徑活化並避免補體系統的過度活化所造成的疾病。C1-inh由478氨基酸組成,具有2個區域: 羧基端區(serpin domain)和氨基端區,其焦點已由早期的血管神經性水腫至目前發現與癌症有關。所以此次的研究目的是探討C1-inh是否對於乳癌細胞會造成增生與遷移的影響。本研究中將已重組好的重組基因於大腸桿菌BL21品系表達出重組蛋白,並且用親和性管柱純化出重組蛋白,利用純化的重組C1-inh測試於人類乳癌細胞(MDA-MB-435S)增生與遷移實驗並觀察其影響。在乳癌細胞增生實驗結果顯示, C1-inh有意義的促進乳癌細胞增生,且當濃度愈高,培養時間愈長,其顯著促進增生的效果就更明顯。而在乳癌細胞遷移實驗結果中C1-inh也是有意義的促進乳癌細胞的遷移,且當濃度愈高,培養時間愈長,其顯著促進遷移的效果更明顯,所以本實驗說明C1-inh確實會促進乳癌細胞增生與遷移的影響。
Abstract
C1-esterase inhibitor (C1-inh) can inhibitor the first complement protein as C1s and C1r activity to reach adjust classical pathway, avoid excessive activation of the complement system to cause disease. C1-inhibitor protein composed of 478 amino acids with two domains: C terminal domain (serpin domain) and N terminal domain. The early focus has been to angioedema associated with cancer found so far. So the purpose of the study was to investigate whether the C1-inh cause for the breast cancer cell proliferation and migration. We use recombinant gene transform Escherichia coli strain BL21(DE3) and expression. Recombinant protein was purified using affinity column. Influence of proliferation and migration on breast cancer cells were tested by purified recombinant C1-inh. In breast cancer cell proliferation results showed, C1-inh significant proliferation of breast cancer, and when the higher concentration, the longer the incubation time, the remarkable effect of promoting proliferation is even more obvious. The results in breast cancer cell migration is also significant in the C1-inh to promote breast cancer cell migration, and when the higher concentration of the longer incubation time, the significant increased migration is more effective. Therefore, this study does note C1-inh to promote breast cancer cell proliferation and migration.
目次 Table of Contents
中文摘要……………………………………………………………….Ⅰ
英文摘要…………………………………………………………………………..Ⅱ
英文縮寫表………………………………………………………………………..Ⅲ
第一章概論
1.1 C1酯酶抑制蛋白的作用…………………………………………………….1
1.2 C1酯酶抑制蛋白的蛋白質結構與氨基端的結構分析…………………….2
1.3C1酯酶抑制蛋白與絲胺酸蛋白酶抑制蛋白(Serpin)家族的關係…………..3
1.4 C1酯酶抑制蛋白化學特性………………………………………………….5
1.5C1酯酶抑制蛋白基因結構……………………………………………………5
1.6 C1酯酶抑制蛋白缺乏與相關性疾病……………………………………….7
1.7 C1酯酶抑制蛋白抑制補體C1的機轉……………………………………..9
1.8 C1酯酶抑制蛋白在免疫學方面的研究……………………………………..9
1.9 C1酯酶抑制蛋白氨基端角色………………………………………………10
1.10研究目的…………………………………………………………………....11
第二章 材料與方法
2.1 分子生物學方法
2.1.1 勝任細胞的製備………………………………………..............................14
2.1.2 轉化作用…………………………………………………………………..15
2.1.3 質體的純化………………………………………………………………15
2.1.4 限制酶切割………………………………………………………………16
2.1.5 再一次的勝任細胞製備、轉化和質體的純化、酶切…………………16
2.2蛋白質分析方法
2.2.1 細菌的培養及基因表達的誘導………………………………………….16
2.2.2 融合蛋白的親和層析表達……………………………………………….17
2.2.3 測量蛋白質液的吸光值………………………………………………….17
2.2.4 對鈉十二烷基的硫酸鹽聚丙烯酰胺凝膠電泳法……………………….18
2.2.5 西方墨點法……………………………………………………………….18
2.2.6 蛋白質透析……………………………………………………………….19
2.2.7 測量蛋白質濃度………………………………………………………….20
2.3 細胞培養及分析方法
2.3.1 人類乳癌細胞株之生長與培養………………………………………….20
2.3.2 細胞增生分析…………………………………………………………….21
2.3.3 細胞遷移分析…………………………………………………………….22
2.3.4 統計分析………………………………………………………………….22
第三章 結果
3.1 表達載體在大腸桿菌株JM 109的接合反應………………………………23
3.2 表達載體在大腸桿菌株BL21的接合反應……………………………….23
3.3 重組蛋白的純化……………………………………………………………23
3.4 西方墨點法…………………………………………………………………24
3.5 蛋白質透析…………………………………………………………………24
3.6 測量蛋白質濃度……………………………………………………………24
3.7 GST-C1-inh蛋白對乳癌細胞MDA-MB-435s增生與遷移的影響
3.7.1 細胞增生結果…………………………………………………………….25
3.7.2 細胞遷移分析結果……………………………………………………….26
第四章 討論…………………………………………………………………….29
參考文獻…………………………………………………………………………33
圖1、在大腸桿菌JM109品系內,1%瓊脂凝膠電泳分析重組基因的限制酶片段…………………………………………………………………………………46
圖2、在表達菌株大腸桿菌BL21內,1%瓊脂凝膠電泳分析重組基因的限制酶片段…………………………………………………………………………………47
圖3、未透析前的重組蛋白質(GST-C1-inh)之SDS-PAGE電泳分析圖……….48
圖4、西方墨點法(Western blot)分析圖……………………………………..49
圖5、未透析前的重組蛋白質(GST-C1-inh)與透析後的重組蛋白質之SDS-PAGE電泳分析比較圖……………………………………………………………………….50
圖6、Multi Gauge V2.02測量蛋白質濃度……………………………………..51
圖7、各種濃度的GST測試乳癌細胞增生分析結果………………………….52
圖8、C1-inh 1μM測試乳癌細胞增生分析結果……………………………….53
圖9、C1-inh 2μM測試乳癌細胞增生分析結果……………………………….54
圖10、C1-inh 4μM測試乳癌細胞增生分析結果……………………………...55
圖11、C1-inh 8μM測試乳癌細胞增生分析結果……………………………...56
圖12、各種濃度的C1-inh測試乳癌細胞增生分析結果……………………...57
圖13、各種濃度的GST測試乳癌細胞遷移分析結果………………………..58
圖14、C1-inh 1μM測試乳癌細胞遷移分析結果……………………………..59
圖15、C1-inh 2μM測試乳癌細胞遷移分析結果……………………………..60
圖16、C1-inh 4μM測試乳癌細胞遷移分析結果……………………………..61
圖17、C1-inh 8μM測試乳癌細胞遷移分析結果……………………………..62
圖18、各種濃度的C1-inh測試乳癌細胞遷移分析結果……………………..63
附圖一 補體活化路徑圖解……………………………………………………..64
附圖二 乙醯鍵結………………………………………………………………..65
附圖三 血漿中的C1酯酶抑制蛋白(C1-inh)結構圖………………………66
附圖四 serpin作用機制………………………………………………………...67
附圖五 serpin domain 結構圖………………………………………………….68
附圖六 C1 -inh核苷酸與氨基酸序列…………………………………………69
附圖七 C1-inh基因外顯子(exon)組成圖……………………………………..70
附圖八 C1-inh絲氨酸蛋白酶抑制蛋白區(C1-inh serpin domain)結構圖..71
附圖九 大腸桿菌表達的C1 -inh建構圖譜……………………………………72
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