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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳彥榮(Yen-Rong Chen) | |
| dc.contributor.author | Shin-Wei Chen | en |
| dc.contributor.author | 陳欣蔚 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:48:25Z | - |
| dc.date.copyright | 2013-03-15 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-02-18 | |
| dc.identifier.citation | Aird, W.C. (2007). Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circulation research 100, 158-173.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15593 | - |
| dc.description.abstract | 甲型血友病是最為常見的血友病類型，由於第八凝血因子 (FVIII) 的缺乏所造成。FVIII 是一種凝血因子，它的變異會使血纖維無法形成血栓，導致無法凝血而血流不止的情形。肝臟為體內 FVIII的主要來源，而肝臟中組成血管內皮之肝竇內皮細胞（liver sinusoidal endothelium cell, LSEC）是體內最主要的 FVIII 生產細胞。過去曾有研究指出，將具正常小鼠之肝竇內皮細胞移植入第八因子缺損的血友病模式小鼠中可以減緩血友病的病症。因此，利用肝竇內皮細胞被期待成為血友病細胞療法之細胞來源之一。在本研究中，分成人類誘導式多能幹細胞株（human induced pluripotent stem cell, hiPSC）的建立以及將 hiPSC 誘導分化為表現 FVIII 的 LSEC 兩部分。
在第一部分中，首先先對人類體細胞株 Hs68 進行再程序化誘導，以慢病毒感染，使之表現多能性相關轉錄因子 c-Myc、Klf4、Oct4 和 Sox2，預期形成人類誘導式多能幹細胞，但在經細胞表面抗原及 mRNA 表現分析後，卻發現細胞並無表現完整的多能性特性。幾經調整重複未果，後直接以 hiPSC 細胞株 253G1 進行 Lsec 的分化研究。 肝竇內皮細胞為特化的內皮細胞，在胚胎發育初期由血管內皮前驅細胞延伸進入初萌發的肝臟後，逐漸分化形成。因此在本研究第二部分以人類胚胎幹細胞分化至血管內皮細胞為基礎，合併抑制 TGFβ 訊息傳導可以誘導小鼠肝竇內皮細胞分化之過去研究，設計出以 BMP4、ActivinA、bFGF、VEFG 和 TGFβ 受體抑制劑（Tbr1ki2）等條件分化培養基（conditional medium）。利用人類iPSC 253G1 細胞株，以胚胎小體 (embryonic body, EB) 培養的方式，誘導分化成肝竇內皮細胞。在經過 16天之分化後，約有 55% 之分化細胞表現肝竇內皮細胞特性，即具透明質酸清除受體 stabilin-2 的表現，且擁有將透明質酸吞噬清除的能力，並具有 FVIII 的 mRNA 和蛋白質表現。 在本篇論文中完成了將人類誘導式多能幹細胞誘導分化至具 FVIII 表現之類肝竇內皮細胞的研究，並期望可用於血友病的治療以及再生醫學之應用。 | zh_TW |
| dc.description.abstract | Hemophilia A is the most common type of hemophilia and is also known as factor VIII (FVIII) deficiency. FVIII is an essential blood clotting factor, and its defects result in the formation of fibrin deficient clots, causing bleeding. liver is a major source of FVIII and the liver sinusoidal endothelial cells (LSEC) are endothelial cells (EC) that line the hepatic microvasculature, sinusoids, which are the major cell type of FVIII production. Previous reports showed that transplantation of LSEC improved the hemophilia phenotype of mice deficient for FVIII. Thus, LSEC is expected to be one of the source for cell therapy of Hemophilia A. My research can be divided into two parts, the generation of human induced pluripotent stem cell (hiPSC) and the differentiation toward LSEC, which can express FVIII, from hiPSC.
In the first part, I infect c-Myc, Klf4, Oct4, and Sox2 to human somatic cell line HS68 by lentivirus for human iPSC formation. However, the results from analysis of cell surface antigen and mRNA expression showed that there is no complete pluripotent-specific expressed in cells. LSEC is a specified EC which emerged into liver bud at early development stage. In my second part of thesis, on the basis of differentiation from human embryonic stem cell (hESC) toward EC, associated the previous study: the inhibition of TGFβ/activin signaling could generate LESC-like from mouse ESC. I designed the protocol for inducing hiPSC toward LSEC through conditional medium with specific cytokines such as BMP4, ActivinA, bFGF, VEGF and TGFβ receptor inhibitor (Tbr1ki2) at different stages. By adopting 253G1 into embryonic body (EB) form, I used the protocol for LSEC induction.16 days later, about 55%-differentiated cells expressed a LESC marker, stabilin-2, a major hyaluronan clearance receptor, and incorporated FITC-labeled hyaluronan. Most importantly, those cells produced FVIII as evidenced by mRNA expression and protein secretion. In conclusion, FVIII-producing LSEC can be derived from human iPS cells and those cells may be an excellent source for cell therapy of hemophilia and the application of regeneration medicine. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:48:25Z (GMT). No. of bitstreams: 1 ntu-102-R99442015-1.pdf: 17853462 bytes, checksum: 36c5ab723a279371f20b900645be6537 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 國立臺灣大學碩士學位論文口試委員會審定書 ii
中文摘要 iii Abstract iv 目錄 vi 第一章 緒論 1 1.1 血友病 (hemophilia) 1 1.2 凝血因子 (coagulation factor) 2 1.3 凝血機制 (coagulation mechanism) 3 1.4 血友病治療 5 1.5 血友病與基因療法 7 1.6 幹細胞 (stem cell) 8 1.7 人類誘導式多能幹細胞 (human induced pluripotent stem cell, hiPSC) 10 1.8 肝竇內皮細胞 (Liver sinusoidal endothelial cell, Lsec) 11 1.9 多能幹細胞至內皮細胞的分化 13 第二章 研究目的與架構 15 第三章 材料與方法 17 2.1 細胞培養 (cell culture) 17 2.1.2 細胞培養液 (culture medium) 17 2.1.3 細胞解凍 17 2.1.4 細胞繼代 (cell subculture) 17 2.1.5 細胞保存 18 2.2 質體建構 (plasmid construction) 18 2.2.1 質體 (plasmid) 18 2.2.2 聚合酶鏈鎖反應 (polymerase chain reaction) 19 2.2.3 勝任細胞製備 (competent cell preparation) 20 2.2.4 細菌轉形 (transformation) 21 2.2.5 限制酶剪切 (restriction enzyme digestion) 21 2.2.6 DNA 片段的回收與萃取 22 2.2.7 DNA黏合 (ligation) 22 2.2.8 小量質體製備 23 2.2.9 大量質體製備 23 2.3 慢病毒製備及感染 (lentivirus production and infection) 24 2.3.1 轉染 (transfection) 24 2.3.2 病毒顆粒收集、濃縮 24 2.3.3 病毒感染 (infection) 24 2.3.4 病毒效價 (titer) 評估 25 2.4 細胞再程序化 (reprogramming) 25 2.4.1 小鼠纖維母細胞 (mouse embryonic fibroblast, MEF) 初代細胞培養 25 2.4.2 餵養細胞 (feeder cells) 準備 25 2.4.3 Hs68 細胞之再程序化誘導 26 2.5 人類胚胎幹細胞/誘導式多能幹細胞培養 26 2.5.1 人類胚胎幹細胞/誘導式多能幹細胞培養液 (ESC/iPSC medium) 26 2.5.2 人類胚胎幹細胞/誘導式多能幹細胞用解離液 26 2.5.3 人類胚胎幹細胞/誘導式多能幹細胞保存液 DAP213 27 2.5.4 人類胚胎幹細胞/誘導式多能幹細胞繼代與培養 27 2.5.5 人類胚胎幹細胞/誘導式多能幹細胞保存 27 2.5.6 人類胚胎幹細胞/誘導式多能幹細胞解凍 27 2.6 人類誘導式多能幹細胞之定性分析 27 2.6.1 鹼性磷酸酶染色 (alkaline phosphatase staining) 28 2.6.2 免疫螢光染色 (immunofluorescence staining assay) 28 2.6.3 mRNA 表現量分析 28 2.7 人類誘導式多能幹細胞分化至肝竇內皮細胞 29 2.7.1 人類誘導式多能幹細胞株 (induced pluripotent cell, iPSC) 29 2.7.2 誘導分化 30 2.8 肝竇內皮細胞定性分析 30 2.8.1 表面受體檢測 30 2.8.2 mRNA 表現量分析 30 2.8.3 清除受體吞噬能力檢測 31 2.8.4 酵素免疫分析法 (Enzyme-linked immunosorbent assay, ELISA) 31 第四章 實驗結果 32 3.1 以慢病毒載體感染體細胞株 32 3.2 以Hs68 再程序化誘導製作誘導式多能幹細胞 32 3.3 類多能幹細胞表達專一性表面抗原 32 3.4 人類誘導式多能幹細胞誘導分化為類肝竇內皮細胞 34 3.5 人類誘導式多能幹細胞抑制 TGFβ 訊息傳導可增進肝竇內皮細胞的特化 34 3.6 類肝竇內皮細胞表現具功能性的清除受體及第八凝血因子 35 第五章 綜合討論與未來方向 37 4.1 類誘導式多能幹細胞的多能性相關基因表現量低 37 4.2 人類體細胞再程序化誘導不完全 37 4.3 類肝竇內皮細胞分化誘導 38 4.4 綜合討論與未來研究方向 39 第五章 圖表 41 圖一、質體 MKOS :: pCDH 41 圖二、慢病毒感染效率分析 42 圖三、Hs68 再程序化誘導流程 43 圖四、Hs68 再程序化誘導48 天後細胞外觀型態 44 圖五、類多能幹細胞表面抗原鹼性磷酸酶表現 45 圖六、類多能幹細胞表面抗原 SSEA4、TRA-1-60、TRA-1-81表現 47 圖七、類多能幹細胞多能性基因表現的定量分析 48 圖八、自人類誘導式多能幹細胞至肝竇內皮細胞之分化誘導流程 49 圖九、經肝竇內皮分化誘導之細胞的階段性分化標記基因表達 50 圖十、經分化誘導肝竇內皮細胞之細胞表面抗原的表現 51 圖十一、類肝竇內皮細胞清除受體對低密度脂蛋白及透明質酸的攝取 52 圖十二、類肝竇內皮細胞的第八凝血因子表現 53 第六章 附錄 54 附錄一、二級凝血級聯反應 54 第七章 參考文獻 55 | |
| dc.language.iso | zh-TW | |
| dc.subject | TGFβ 受體抑制 | zh_TW |
| dc.subject | 甲型血友病 | zh_TW |
| dc.subject | 肝竇內皮細胞 | zh_TW |
| dc.subject | 誘導式多能幹細胞 | zh_TW |
| dc.subject | 第八凝血因子 | zh_TW |
| dc.subject | induced pluripotent stem cell | en |
| dc.subject | factor VIII | en |
| dc.subject | liver sinusoidal endothelial cell | en |
| dc.subject | TGFβ receptor inhibitor | en |
| dc.subject | hemophilia A | en |
| dc.title | 以人類多能幹細胞分化至肝竇內皮細胞之研究 | zh_TW |
| dc.title | The differentiation of liver sinusoidal endothelium cells from human induced pluripotent stem cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李昆達(Kung-Ta Lee),黃兆祺(Eric Hwang) | |
| dc.subject.keyword | 誘導式多能幹細胞,肝竇內皮細胞,第八凝血因子,TGFβ 受體抑制,甲型血友病, | zh_TW |
| dc.subject.keyword | induced pluripotent stem cell,liver sinusoidal endothelial cell,factor VIII,TGFβ receptor inhibitor,hemophilia A, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-02-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| Appears in Collections: | 生化科技學系 | |
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| ntu-102-1.pdf Restricted Access | 17.44 MB | Adobe PDF |
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