補體第一酯酶抑制蛋白(C1 inhibitor, C1 INH)是絲胺酸蛋白酶抑制蛋白(serine protease inhibitor, serpin))家族的成員之一，C1 INH是唯一能夠抑制C1分子次單元C1r和C1s這二個絲胺酸蛋白酶的蛋白質。C1 INH 是單胜肽鏈的蛋白質，高度醣基化，完整的C1 INH分子量大約是105KD，全長有478個胺基酸，有13個已知的醣基化位點，其中10個已知醣基化位點和7個有可能的醣基化位點位於前98個胺基酸的N端序列之中。許多種類的醣類分子位癌細胞的表面和細胞外基質 (extracellular matrix, ECM)，細胞表面的醣類分子調節癌細胞的生理反應，包括生長、黏附、轉移等。醣類分子同時也可以和生長因子等配位體結合，調節配位體和接受器的結合以及接下來的訊息傳導反應。如果可以影響這些醣類分子的作用，或許可以抑制細胞訊息的傳導，抑制癌細胞癌細胞的生理反應，成為處理癌症一個有效的方法。我們將編碼為C1 INH N端98個胺基酸序列(C1 INH NT98)的基因接到表達載體pGEX-2T上，以大腸桿菌(BL21品系)表達出這段重組蛋白質，接下來測試這段重組蛋白質對乳癌細MDA-MB-435s生長及轉移的影響。在對細胞增生(cell proliferation)的實驗中我們發現，以原核生物表達出來的未經過醣基化C1 INH NT98加到細胞培養液中和純粹只以培養液培養的細胞比較，C1 INH NT98抑制乳癌細胞的生長，而在細胞遷移(cell migration)實驗結果也顯示這段重組蛋白抑制了乳癌細胞的遷移，所以缺少醣基化的C1 INH蛋白 N端序列可以抑制乳癌細胞，而其中如何抑制的機制則需要更進一步的研究。

C1 inhibitor (C1 INH) is a member of the serine protease inhibitor (serpin) superfamily. It is the only physiological inhibitor of protease C1r
and C1s in the complement system. C1 INH is a single chain glycoprotein with apparent molecular weight of 105 KDa, consisting of 478 amino acids. C1 INH N-terminal domain includes first 98 amino acids with 10 definite and 7 potential glycosylation site. Various of carbohydrates are present on the cell surface and component of ECM (extracellular matrix) in every eukaryotic cell, including both cancer cells and cells that are important for tumur survival. Carbohydrates on the cancer cell surface have been shown to be important in many aspects of cancer cell physiological processes, involved in cell growth and cell adhesion.
Carbohydrates are also able to bind and interact with growth factors and other proteins that trigger signal transduction. Interfere carbohydrates maybe offer a useful therapeutic approach for treating cancers. In order to understand whether the C1 INH NT98 polypeptides can influences cancer or not, we amplified a DNA fragment encoding C1 INH N-terminal domain 98 residues (C1 INH NT98) by PCR, and transfer to the plasmid pGEX-2T, than use E.coli (BL21 strain) to express the non-glycosylated polypeptides, and further analyze the influence of the effective roles exhibited by the polypeptides non-glycosylated on breast cancer cell MDA-MB-435s. Proliferation and migration assays in our experiment showed that non-glycosylated C1 INH NT98 can inhibited breast cancer cell growth and migration, and the mechanism needed to be clarified clearly through extensive research.

中文摘要------------------------------------------------------------1
英文摘要------------------------------------------------------------2
第一章 背景
1.1 補體蛋白C1 抑制劑(C1 inhibitor，C1 INH)的生理功能 ---------------3
1.2 C1 INH 的結構----------------------------------------------------4
1.3 C1 INH SEPIN 區域的抑制功能--------------------------------------5
1.4 C1 INH 缺陷的遺傳疾病--------------------------------------------6
1.5 C1 INH 基因突變所導致的缺失-------------------------------------7
1.6 C1 INH 的氨基末端(N-terminal domain)------------------------------9
1.7 研究目的-------------------------------------------------------11
第二章 材料與方法
實驗材料來源-------------------------------------------------------12
實驗方法-----------------------------------------------------------13
2.1 C1 INH NT98 基因工程改造
2.1.1 聚合酶鏈反應引子的設計--------------------------------------13
2.1.2 聚合酶鏈鎖（PCR）反應---------------------------------------14
2.1.3 瓊脂糖凝膠電泳分析------------------------------------------14
2.1.4 TA cloning--------------------------------------------------14
2.1.5 勝任細胞的 (competent cell) 製備----------------------------15
2.1.6 轉化作用(Transformation)------------------------------------15
2.1.7 質體的純化--------------------------------------------------15
2.1.8 限制酶切割及質體片段從凝膠中回收----------------------------16
2.1.9 連接反應----------------------------------------------------17
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-----------------------------------17
2.2.2 融合蛋白的親和層析表達---------------------------------------18
2.2.3 測量蛋白質液的吸光值-----------------------------------------18
2.2.4 SDS-聚丙烯醯胺膠體電泳 (SDS-PAGE)----------------------------18
2.2.5 蛋白質透析---------------------------------------------------19
2.3 細胞培養及分析方法
2.3.1 人類乳癌細胞株（MDA-MB 435s）之生長與培養-------------------19
2.3.2 細胞增生分析 (cell proliferation assay) --------------------20
2.3.3 細胞遷移分析(cell migration assay)--------------------------20
2.3.4 統計分析----------------------------------------------------21
第三章 結果
3.1 C1 INH NT98 基因的重組-----------------------------------------22
3.2 GST- C1 INH NT98 蛋白質的表達及SDS-PAGE 分析-------------------22
3.3 GST-C1 INH NT98 蛋白對乳癌細胞MDA-MB-435s 生長細胞增生結果-----23
3.4 GST-C1 INH NT98 蛋白對乳癌細胞MDA-MB-435s 細胞遷移的影響結果---24
第四章 討論--------------------------------------------------------25
參考文獻-----------------------------------------------------------27

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