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研究生:葉慈馨
研究生(外文):Chi-xin Ye
論文名稱:重組C1-inhibitor對人類乳癌細胞生長及遷移的影響
論文名稱(外文):Influence of Recombination C1-Inhibitor on proliferation and migration of Human Breast Cancer Cells
指導教授:何世屏
指導教授(外文):Shiping He
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:67
中文關鍵詞:增生轉移
外文關鍵詞:proliferationmigration
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人類補體C1抑制劑(C1 inhibitor)屬於絲氨酸蛋白酶抑制劑家族的成員。補體C1抑制劑是生理上C1s和C1r唯一具有生理意義的抑制劑。補體C1抑制劑在典型補體路徑上扮演著必要性的角色。補體C1抑制劑為血漿活化蛋白釋放酶(plasma kallikrein)和激肽釋放酶(kinin)系統內活化factor XII的主要抑制劑中的一個。有證據證明補體C1抑制劑也可抑制凝集素(lectin)途徑中的蛋白酶、替代補體路徑中的C3b、胞漿素活化劑 (plasminogen activator) 、活化factor XI, 和活化蛋白釋放(kallikrein)。在正常的乳細胞內補體C1抑制劑基因的表達有向下調節的傾向而惡性細胞的基因表達具顯著上升。因此本研究的主要目的在於研究重組補體C1抑制劑379到478片段對於人類乳癌細胞是否有任何的影響。在此本研究中我們建構了補體C1抑制劑379到478片段於質體上。將建構好的質體轉移到大腸桿菌BL21(DE3)品系內來表現此蛋白且使用榖胱甘肽轉移酶(GST)親和層析法來純化此蛋白。使用由大腸桿菌內純化好的補體C1抑制劑來研究是否此蛋白對於人類乳癌細胞(MDA MB 435s)的增生和轉移有任何的影響。結果顯示1到100 nM濃度的重組補體C1抑制劑379到478片段在4、6或8天可抑制MDA MB 435s細胞的增生。然而,該重組蛋白的濃度上升到100 nM時,培養48小時之後才顯示出對癌細胞轉移的顯著效應。實驗結果顯示重組補體C1抑制劑379到478片段對於MDA MB 435s細胞的增生和轉移均有一定的抑制效果。
Human C1 inhibitor (C1 INH) belongs to the superfamily of serine protease inhibitors (serpins). It is the only known physiological inhibitor of C1s and C1r, therefore C1 INH plays an essential role as a regulator of complement classical pathway. C1 INH is also one of the major inhibitors of plasma kallikrein and activated factor XII of the kinin system. There is evidence that C1 INH can also inhibit the complement lectin pathway proteases, and the complement alternative pathway (C3b), plasminogen activator, activated factor XI, and kallikrein. A recent report indicated C1 inhibitor was significantly down-regulated in normal breast cells compared to malignant cell line. Thus, the major aim of this project is to study the effect of recombinant C1 inhibitor Ile379-Ala478 on human breast cancer cells. In this study, we constructed plasmid containing DNA fragment for C1 inhibitor C-terminal negion of Ile379-Ala478. Then the constructed plasmid was transferred into E.coli BL21(DE3) for expression. The recombinant protein was purifed by GST-affinity chromatography. Recombinant C1 inhibitor produced by E.coli was used to treat breast cancer cells (MDA MB 435s) to study its effect on cell proliferation and migration. Results showed that recombinant C1 inhibitor Ile379-Ala478 protein inhibited proliferation of MDA MB 435s cells after four, six or eight days, incubation in the presence of recombinant protein (1~100 nM). However, cell migration was inhibited only at 100 nM on 48 hrurs incubation..Our experimental data showed that the recombinant C1 inhibitor Ile379-Ala478 inhibited proliferation and migration of MDA MB 435s cells.
Contents
Abstract •••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1
Abstract (in Chinese) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2
Abbreviation • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •3
Chapter 1 Introduction• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •4
Chapter 2 Materials and methods•••• • • • • • • • • • • • • • • • • • • • • • • • • 14
2.1 Materials••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 14
2.2 Approaches in molecular biology • • • • • • • • • • • • • • • • • • • • • • • • 17
2.2.1 Preparation of template DNA fragment for PCR • • • • • • • • • • • • • 17
2.2.2 Amplification of C1 inhibitor Ile379-Ala478 gene by PCR•• • • • • •••17
2.2.3 T-A cloning ••••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •18
2.2.4 Transformation of intermediate plasmid••• • • • • • • • • • • • • • • • • •19
2.2.5 Purification of plasmids and checking••••••• • • • • • • • • • • • • • • • •20
2.2.6 Ligation of pGEX-2T and C1 inhibitor Ile379-Ala478••••• • • • • • • 20
2.2.7 Checking the plasmid of pGEX-2T and C1 inhibitor Ile379-Ala478•21
2.3 Approaches in recombination protein detection ••• • • • • • • • • • • • • •21
2.3.1 Expression and purification of C1 inhibitor Ile379-Ala478 ••••• • • •21
2.3.2 Sodium dodecylsulfate-polyacryamide gel electrophoresis ••• • • • • 22
2.4 Approaches in cell biology •• • • • • • • • • • • • • • • • • • • • • • • • • • • •22
2.4.1 Cells and culture conditions ••• • • • • • • • • • • • • • • • • • • • • • • • • 22
2.4.2 In vitro cell proliferation assay• • • • • • • • • • • • • • • • • • • • • • • • •22
2.4.3 In vitro wound healing assay •••••• • • • • • • • • • • • • • • • • • • • • • •23
Chapter 3 Results••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25
3.1 Polymerase chain reaction amplification and PCR cloning •••• • • • • •25
3.2 Expression and purification of C1 inhibitor Ile379-Ala478 •• • • • • • •25
3.3 In vitro cell proliferation assay •• • • • • • • • • • • • • • • • • • • • • • • • • •26
3.4 In vitro wound healing assay• • • • • • • • • • • • • • • • • • • • • • • • • • • • 27
Chapter 4 Discussion • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 29
Reference • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •33



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