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系統識別號 U0026-0708201214265600
論文名稱(中文) 利用馬來腹蛇蛇毒蛋白作為架構設計針對整合蛋白αvβx與α5β1之專一性拮抗物
論文名稱(英文) Development of Integrins αvβx and α5β1-specific Antagonists Using Rhodostomin as a Scaffold
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 詹秉澤
研究生(英文) Ping-Tse Chan
學號 s16991087
學位類別 碩士
語文別 中文
論文頁數 119頁
口試委員 指導教授-莊偉哲
口試委員-羅玉枝
召集委員-王淑鶯
中文關鍵字 馬來蝮蛇蛇毒  整合蛋白  黑色素瘤  血管新生 
英文關鍵字 Rhodostomin  integrin  melanoma  angiogenesis 
學科別分類
中文摘要 整合蛋白是由α次單元及β次單元所組成的穿膜蛋白,整合蛋白參與了許多生理機制,包括凝血作用、生長、血管新生與感染。先前的研究中也指出整合蛋白與許多疾病息息相關,包括癌症。說明了整合蛋白作為癌症治療中藥物設計目標的可能。去整合蛋白指的是一群在蛇毒中被發現由47到84個胺基酸及4到7對雙硫鍵所組成的蛋白,具有與整合蛋白結合能力的拮抗物。這群去整合蛋白主要是經由RGD所形成的結構與整合蛋白進行結合的,除了RGD motif外,RGD鄰近的部分與其C端均指出對於整合蛋白的辨認上具有重要性。在本篇研究當中,將去整蛋白Rhodostomin (Rho)作為藥物設計的架構,Rho源自於馬來蝮蛇具有68個胺基酸、6對雙硫鍵及RGD構型,我將使用Rhodostomin作為基本架構,設計出對於整合蛋白αvβx與α5β1具專一性的拮抗物,希望可發展出抵抗癌症的前驅藥物。除此之外,為了得到更多未來在設計拮抗物的資訊,本篇論文中也將對RGD鄰近的N端及C端進行研究。我已成功表現並純化出17個Rhodostomin突變物,發現了其中一個稱為39KKARTICARGRGDNP-YH68(簡稱為KG)的Rhodostomin 突變物對於整合蛋白αvβx與α5β1同時具結合能力,此蛋白與整合蛋白αvβ3、αvβ5、αvβ6、α5β1與αIIbβ3的IC50分別為22.2、73.3、62.5、10.6與31782.8 nM。另外在RGD motif鄰近N端部位Rho 46XXPRGDMP53的探討,發現突變為負電胺基酸46DDPRGDMP53和46EEPRGDMP53後,發現對於αIIbβ3和α5β1的結合能力有顯著變差。接著在RGD motif鄰近C端部位Rho 48PRGDMPXX55的探討,將54、55位置的氨基酸分別突變為各類胺基酸A、K、R、N與Y,大部分的突變對整合蛋白的辨識沒有影響,最有趣的部份是Rho PRGDMP54YY55 ,對於αvβ3、α5β1與αIIbβ3合能力的IC50為4.9、69.6和58.0 nM顯示了對於整合蛋白α5β1的結合能力有 3.7倍的上升,更細部的探討中顯示Rho D54Y才是對於整合蛋白α5β1結合能力上升的主因(IC50為81.7 nM)。研究結果中最有意義的蛋白39KKARTICARGRGDNP-YH68,可辨認專一性整合蛋白αvβx與α5β1,而在黑色素瘤的發展過程中,整合蛋白αvβ3、αvβ5與α5β1表現量會逐漸上升,於是將KG進行In Vitro實驗,發現KG可抑制黑色素瘤細胞A375的生長、移動、侵入及附著,在使用HUVEC進行血管新生的實驗中也發現KG可有效抑制血管新生。在In vivo的實驗中,證明了KG可有效抑制B16F10(小鼠黑色素瘤細胞)在B6老鼠體內生長,更進一步的使用NOD SCID免疫缺失小鼠移植A375人類黑色素瘤細胞後使用KG進行治療,發現可顯著抑制A375在NOD SCID免疫缺失小鼠上生長。這些結果說明了KG對於黑色素瘤的治療上,將會是一個很有潛力的藥物候選。
英文摘要 Integrins are α/β heterodimeric transmembrane proteins. With their involvement in many biological functions, such as thrombosis, growth, angiogenesis, and inflammation, integrins are discovered to associate with a broad range of diseases, including cancer and immunodeficiency disorders. Thus, integrins could serve as potential therapeutic targets in treating many diseases. Disintegrins are a family of potent integrin inhibitors found in snake venoms that contain 47 to 84 amino acids with 4-7 disulfide bonds. It has been shown that the RGD motif, the regions adjacent to the RGD motif, and C-terminal region of disintegrins are important for their activity and selectivity. In this study I used rhodostomin (Rho) as protein scaffold to design dual integrins αVβx and α5β1-specific disintegrin and to study the effect of the regions adjacent to the RGD motif. Rho is a snake venom protein isolated from Calloselasma rhodostoma and contains 68 amino acid residues with a PRGDMP motif. I have expressed seventeen Rho mutant proteins and purified them to homogeneity. I designed a Rho mutant containing 39KKARTICARGRGDNP53 amino acid sequence, which can selectively inhibit integrins αvβx and α5β1. It inhibited integrins αvβ3, αvβ5, αvβ6, α5β1, and αIIbβ3 with the IC50 values of 22.2, 73.3, 62.5, 10.6, and 31782.8 nM, suggesting that it is a dual integrins αVβx and α5β1-specific mutant. According to the cell adhesion analysis of Rho 46XXPRGDMP53mutants, such as 46DDPRGDMP and 46EEPRGDMP, it showed that the N-terminal negatively residues adjacent to the RGD motif decreased their activity to integrins αIIbβ3 and α5β1. In contrast, the C-terminal charge residues adjacent to the RGD motif caused little effect on their activity based on the cell adhesion and platelet aggregation analyses of Rho PRGDMP53XX54 mutants. Interestingly, Rho PRGDMP54YY55 mutant inhibited integrins αvβ3, α5β1, and αIIbβ3 with the IC50 values of 4.9, 33.6, and 58.0 nM, showing a 7.6-fold increase in inhibiting integrin α5β1. We also found that Rho PRGDMP54DY55mutant exhibited higher integrin α5β1 binding affinity. It is known that the progression of melanoma is associated with the expression of integrins αvβ3 and α5β1. We found that integrins αvβx and α5β1-specific mutant and Rho inhibited the adhesion, migration, and invasion of human A375 melanoma cells. It also significantly suppressed tumor growth in C57BL/6 mice implanted with B16F10 melanoma cells and in NOD SCID mice implanted with A375 human melanoma cells. The results of this study will serve as the basis to design potent integrins-specific antagonists for melanoma.
論文目次 中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
表目錄 VIII
圖目錄 IX
縮寫檢索表 XI
儀器 XII

第1章緒論
1-1 背景資料 1
1-2 整合蛋白(integrin)的介紹 1
1-3 癌症與整合蛋白的關係 3
1-4 整合蛋白在癌症誘導之血管新生中扮演的角色 4
1-5 去整合蛋白與馬來蝮蛇去整合蛋白的介紹 5
1-6 研究的動機與目標 6
第2章 材料與方法
2-1 Rhodostomin與突變株重組蛋白的製備 9
2-1-1 基因重組及蛋白表現之材料與方法 9
2-1-2 Rhodostomin重組蛋白基因之構築 11
2-1-3 Rho重組蛋白的表現與純化 18
2-1-4 Rhdostomin重組蛋白之質譜鑑定 23
2-2 Rhodostomin與其突變株抑制血小板凝集之研究 24
2-2-1 血小板之製備 24
2-2-2 Rhodostomin突變蛋白對於血小板凝集抑制之分析 25
2-3 細胞株及培養方法 25
2-3-1 細胞培養 25
2-4 Rho與Rho突變重組蛋白抑制細胞黏著之研究 28
2-4-1 Fibronectin之製備 29
2-4-2 Rho突變重組蛋白抑制細胞黏著能力之分析 31
2-5 檢測細胞表面之整合蛋白表現量 32
2-6 Rho突變重組蛋白抑制A375黑色素瘤細胞移動及侵犯能力研究 34
2-7 Rho突變重組蛋白抑制A375黑色素瘤細胞生長之研究 36
2-8 Rho突變重組蛋白抑制A375黑色素瘤細胞形成群落之研究 37
2-8-1 Colony formation assay (Anchorage-dependent) 37
2-8-2 Colony formation assay (Anchorage-independent) 38
2-9 Rho突變重組蛋白對於血管新生影響之研究 39
第3章 結果
3-1 Rhodostomin與突變株重組蛋白之備製與鑑定 41
3-2 Rho突變株抑制血小板凝集的結果 42
3-3 Rho突變株抑制細胞黏著的結果 43
3-3-1 Rho突變株對於整合蛋白v3之結合能力之結果 43
3-3-2 Rho突變株對於整合蛋白51結合能力之結果 44
3-3-3 Rho突變株對於整合蛋白IIb3結合能力之結果 45
3-3-4 Rho突變株中較有應用性者之數據整理 45
3-3-5 Rho突變株對於整合蛋白v5的結合能力之結果 47
3-3-6 Rho突變株對於整合蛋白v6結合能力之結果 47
3-4 Rho突變株對於黑色素細胞瘤的影響 47
3-4-1 流式細胞儀檢測黑色素瘤之整合蛋白表現量 49
3-4-2 Rho突變蛋白株抑制黑色素瘤黏著的結果 49
3-4-3 Rho突變蛋白株抑制黑色素瘤移動的結果 50
3-4-4 Rho突變蛋白株抑制黑色素瘤侵犯的結果 50
3-4-5 Rho突變蛋白株抑制黑色素瘤侵犯的結果 51
3-4-6 Rho突變蛋白株抑制黑色素瘤生長的結果 51
3-4-7 Rho突變蛋白株抑制黑色素瘤在懸浮環境形成群落的結果 52
3-5 Rho及各Rho突變株對於血管新生影響之結果 52
3-5-1 流式細胞儀檢測HUVEC之整合蛋白表現量 53
3-5-2 Rho突變蛋白株抑制HUVEC生長的結果 53
3-5-3 Rho突變蛋白株抑制血管新生的結果 54
3-6 Rho突變株KG動物實驗之結果 54
3-6-1 KG對於C57BL/6小鼠中B16F10老鼠黑色素細胞瘤生長的抑制結果 54
3-6-2 KG對於NOD SCID免疫缺失小鼠中A375人類黑色素細胞瘤生長的抑制結果 55
3-7 Rho與Rho突變株對於HT-29的影響 55
3-7-1 HT-29表面整合蛋白之表現量 56
3-7-2 Rho及Rho突變株對於HT-29生長之影響 56
第4章 討論
4-1 利用本研究突變株的結果進行既有拮抗物的改良 59
4-2 KG對於內皮細胞作用機制討論 60
4-3 動物實驗未來可行的方向 61
4-4 多目標拮抗物的優勢與劣勢 62
4-5 共同抑制整合蛋白與生長因子受體下游路徑的藥物發展趨勢 63
第5章 結論 65
參考文獻 68
表 71
圖 79
附錄 110
自述 119
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