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研究生:林佳霓
研究生(外文):Chia-Ni Lin
論文名稱:探討突變型人類凝血第九因子的功能
論文名稱(外文):Functional Characterization of Mutant Human Coagulation factor IX
指導教授:林淑華林淑華引用關係
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:96
中文關鍵詞:B型血友病凝血第九因子凝血因子基因治療小鼠模型
外文關鍵詞:Hemophilia BCoagulation Factor IXCoagulation factorGene therapymouse model
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B型血友病是一種性聯遺傳疾病,主要是因為缺乏具有凝血功能的第九因子造成。B型血友病目前的治療方法是用注射血漿純化的或是合成的第九因子,本論文第一個部份是研發一種具有較高活性的第九因子,以便在血友病的治療方面可以降低血友病病人治療時的使用量及成本。我們製造了一種合成的第九因子Factor IX–Triple。該變種第九因子在純化的系統中具有比野生型高13倍的活性,這樣的第九因子具有三個單點突變,分別位於胺基酸序列86,277及338的位置。而且它與活化的第八因子的親和力也比野生型高十倍左右。把這樣的蛋白注射到血友病小鼠的體內評估該蛋白的治療狀況,可以發現這種蛋白質在體內的半衰期和野生型第九因子相差不多。更重要的是該蛋白的活性在小鼠體內仍然可以表現出比野生型高出七倍的活性,明顯可以應用於治療血友病,而且其效能遠比野生型好。我們另外也用了水流體動力學的原理透過尾靜脈注射的方式,將可以在肝內專一性表現第九因子的DNA打入血友病小鼠體內,同樣也可觀察到FIX-Triple具有比野生型高3.5倍的專一活性。另外我們也用帶有不同突變第九因子基因的第八血清型AAV(serotype 8 recombinant adeno-associated virus)病毒由尾靜脈注射入小鼠體內,同樣也可看到Factor IX–Triple的專一活性比野生型高出七倍。我們用了三種不同劑量,可以看到第九因子表現的活性及濃度會隨著打入的劑量而改變,但是就其專一活性而言,可以觀察到打入factor IX-Triple的小鼠是打野生型的七倍。當打入最低劑量時,factor IX-Triple可以使血友病小鼠平均達到正常血漿35%的活性,已趨近正常範圍,反觀打入野生型第九因子的血友病小鼠,只有達到正常血漿6%的活性,相當於中度血友病的程度。總結我們產生了一個比野生型具有更高的活性的第九因子,在將來或許可以取代現有治療用蛋白而降低血友病治療的成本及風險。
人類凝血第九因子的重鍊是含有酵素功能的區域。本論文第二個部分是針對在重鍊區內兩個特定胺基酸發生突變的病人FIX FuChou (G190V),FIX Nan Tou 1(F192V)做研究,試圖找出致病機轉。利用血友病小鼠模型以水流體動力學原理自尾靜脈分別打入可以表現野生型及此兩種突變型第九因子的質體。觀察到這兩種蛋白的表現量比野生型略少。由aPTT測得的凝血活性則有很大差異,野生型為0.60 U/mL, G190V及F192V分別是0.08 U/mL 及 0.14 U/mL。計算其專一凝血活性後,野生型為244.14 U/mg,G190V及F192V分別是50.01 及64.62 U/mg。但是藉由肝臟的埋臘切片染色觀察,其表現量並無明顯差異,所以推論這兩個病人血中第九因子濃度偏低的主要原因可能不是蛋白質合成的過程有問題。同時藉由體外表達第九因子分析其功能,發現純化後的G190V 及F192V蛋白活性分別是野生型第九因子的12.2%及16.8%。同時也發現比野生型第九因子更容易被降解。當他們被活化的第十一因子活化時,比正常的第九因子多了幾個被切割的位置。經由胺基酸序列分析可以確定切點是Arg116-Leu117,Lys265-Tyr266,Arg327-Val328以及 Arg338-Ser339。這些被切的胺基酸位置也是位於第九因子的球狀結構表面。同時G190V也較容易被纖維蛋白酵素降解。配合蛋白結晶繞射的電腦模擬圖結果,推論在G190及 F192發生點突變可能造成第九因子被活化後的結構摺疊異常而造成穩定性變差。因此我們推論凝血活性的減低,以及蛋白質穩定性變差是造成病人表現血友病症狀的主要原因。
Hemophilia B is an X-linked inherited disorder caused by the lack of a functional coagulation Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. The first part of this thesis is focused on developing new factor IX for therapeutic use. In an effort to reduce factor usage and cost, we investigated the potential use of human Factor IX (FIX) variants with enhanced specific activity. We generated and purified seven recombinant FIX variants using alanine replacement and assayed their activity in vitro. One variant containing three substitutions (R86A/E277A/R338A, FIX-Triple) exhibited ~13-fold higher specific activity and a 10-fold increased affinity for human Factor VIIIa than FIX-wildtype (FIX-WT) and was thus investigated systematically in vivo. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than FIX-WT. Additionally, liver-specific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific activity compared to FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific activity of FIX-Triple than FIX-WT (35% vs. 6% of normal human plasma intrinsic activity, respectively). In conclusion, we demonstrate the generation of a novel FIX variant with substantially enhanced specific activity in vitro and in vivo, that can be utilized for protein replacement therapy as well as gene-based therapeutic strategies.
The second part of this thesis is focused on two mutant factor IX proteins. The heavy chain (amino acid residues 181-415) of human blood clotting factor IX (FIX) contains the protease function of FIX. Two hemophilia B patients with missense mutations at residues G190 (G25, chymotrypsinogen numbering system) and F192 (F27) exhibited severe and moderate bleeding phenotypes, respectively. The patient with the FIXG190V (glycine replaced by valine) variant had severe bleeding episodes with <1% clotting activity and only 36% of the normal FIX protein levels in plasma. The patient with the F192V variant had only 2% clotting activity and <6% of the normal FIX protein levels in plasma. Hemophilia B mice, which contain an engineered-deletion of FIX gene) expressing human wild type (WT) or the two mutant FIX have been created by hydrodynamic technique that directs the exogenous DNA synthesis in the liver. In this model system a significant difference in the plasma FIX clotting activity was observed (IXWT: 0.60 U/mL, G190V: 0.08 U/mL and F192V: 0.14 U/mL respectively). Since immunohistochemical staining showed similar amounts of FIX in the liver, the synthesis and secretion pathways are less likely to be the major pathogenic mechanisms onderlying the low amount of the mutant variants in the plasma. Decreased specific clotting activities were demonstrated for both the G190V (12.2% of WT FIX) and F192V (16.8% of WT FIX) variants. Purified G190V and F192V proteins were more sensitive to degradation than wild type FIX, especially after being activated by factor XIa. The vulnerable sites were mapped to the peptide bonds at Arg116-Leu117, Lys265-Tyr266, Arg327-Val328, and Arg338-Ser339, conferring the exposed loops of the FIX molecule. The zymogen form of FIX G190V was also easily hydrolyzed by plasmin. Mutations in the G190 and F192 region may result in misfolding and/or decreased stability of FIX in plasma. We conclude that the defects in both the clotting activities and stabilities of G190V and F192V variants of the FIX protein contributed to the clinical phenotypes of these two mutations in the hemophilia B patients.
口試委員會審定書
誌謝 i
中文摘要 ii
ABSTRACT iv
圖目錄 1
表目錄 2
第一章 緒論 3
第二章 研發具有高凝血活性第九因子 13
2.1摘要 13
2.2實驗材料及方法 15
2.3實驗結果 27
2.4實驗討論 31
第三章 探討血友病病人的致病機轉 35
3.1摘要 35
3.2簡介 36
3.3實驗材料及方法 37
3.4實驗結果 44
3.5實驗討論 47
第四章 製造抗第九因子單株抗體 49
第五章 結語及未來展望 52
參考文獻 55
圖 64
表 88
附錄 96
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