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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李心予(Hsinyu Lee) | |
dc.contributor.author | Kai Hsia | en |
dc.contributor.author | 夏凱 | zh_TW |
dc.date.accessioned | 2021-06-17T08:10:13Z | - |
dc.date.available | 2022-08-20 | |
dc.date.copyright | 2019-08-20 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-16 | |
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Seifalian, The use of animal mode | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73786 | - |
dc.description.abstract | 鞘氨醇-1-磷酸(Sphingosine-1-phosphate, S1P)具有促進血管內皮細胞的增殖、遷移的生理作用,因而參與血管新生、保持血管穩定性及通透性。近年有文獻報導, S1P可以保護血管內皮細胞表面的醣蛋白Syndecan-1 (SDC1),抑制其脫落,從而防止血管內凝血的發生。在目前的研究中,我們將人體臍帶中的臍靜脈做去細胞處理,再將臍靜脈血管內皮細胞 (human umbilical vein endothelial cells , HUVEC) 和臍帶血中的血管內皮前趨細胞 (endothelial progenitor cells, EPC)分別種植到去細胞骨架上,比較添加S1P在構建這一組織工程血管的效果。首先染色確定種植細胞的數量和種類,再用血小板在組織工程血管的貼附數量和凝血時間評估S1P抗凝血功能。為進一步探討S1P的抗凝血機制,我們發現S1P對於血管內皮細胞表面的一種醣蛋白SDC1具有保護作用,因此設計一個體外實驗,用消化SDC1的專一性酵素matrix metalloproteinase-7 (MMP-7)將SDC1切除,觀察在添加S1P時,血小板在血管內皮細胞表面貼附數量的變化。以上實驗結果顯示,S1P可以促進HUVEC及EPC在去血管骨架上的貼附及增殖,在種植HUVEC的組織工程血管,S1P具有抗凝血的功效,其原因在於S1P通過保護血管內皮細胞表面的醣蛋白SDC1,防止其脫落,從而達到抗血管內凝血的目的。但在種植EPC的血管中抗凝血的效果並不明顯。
為進一步證明S1P在組織工程血管 (tissue-engineered vascular grafts, TEVGs) 的抗凝血功效,建立同種血管移植的平台,將大鼠的血管內皮細胞種植在去血管骨架上,並植入大鼠主動脈。比較以下五組血管(n=6): (1) 同種大鼠主動脈 (rat allogenic aorta, RAA); (2) 去細胞大鼠主動脈 (decellularized RAA, DRAA); (3) 去細胞大鼠主動脈添加S1P (DRAA with S1P , DRAA/S1P); (4) 種植血管內皮細胞之去細胞大鼠主動脈 (recellularization, DRAA/EC); 及 (5) 種植血管內皮細胞時添加S1P之去細胞大鼠主動脈 (DRAA with S1P and EC recellularization, DRAA/EC/S1P) 在植入動物體內後14天血管之通暢率,以體內(in vivo)實驗證明S1P抗凝血的功效。實驗結果顯示,體外分離培養的大鼠血管內皮細胞可表現表面抗原CD31,並可吞噬特異性染劑Dil-Ac-LDL,具有成熟血管內皮細胞的特性。 S1P同樣可以增加syndecan-1在大鼠血管內皮細胞的表現量。由血管移植的體內實驗結果可見,RAA 和 DRAA/EC/S1P組在術後14天 血管100% 通暢,無血栓形成,病理染色可見其內皮化程度較佳,血管結構較完整,且發炎反應亦不明顯,而DRAA, DRAA/S1P and DRAA/EC組則有血管內凝血的狀況。進一步確認添加S1P所構建的血管有較佳的抗凝血功效。 | zh_TW |
dc.description.abstract | Sphingosine-1-phosphate (S1P) has been known to promote endothelial cell (EC) proliferation and protect Syndecan-1 (SDC1) from shedding, thereby maintaining this antithrombotic signal. In the present study, I investigated the effect of S1P in the construction of a functional tissue-engineered blood vessel by using human endothelial cells and decellularized human umbilical vein (DHUV) scaffolds. Both human umbilical vein endothelial cells (HUVEC) and human cord blood-derived endothelial progenitor cells (EPC) were seeded onto the scaffold with or without the S1P treatment. After the efficacy of re-cellularization was determined, the antithrombotic effect of S1P was examined by the anti-aggregation tests measuring platelet adherence and clotting time. Finally, I altered the expression of SDC1, a major glycocalyx protein on the endothelial cell surface, using matrix metalloproteinase-7 (MMP-7) digestion to explore its role using platelet adhesion tests in vitro. The result showed that S1P enhanced the attachment of HUVEC and EPC and reduced thrombus formation compared to controls. Our results also identified S1P reduced SDC1 shedding from HUVEC responsible for inhibition of platelet adherence. However, no significant antithrombogenic effect of S1P was observed on EPC.
Furthermore, I verified the result by implanting tissue-engineered vascular grafts (TEVGs) in rats which are constructed by decellularized allografts and rat EC with the addition of S1P. The in vivo patency rate and endothelialization for five groups of decellularized vascular grafts (each n = 6) in a rat abdominal aorta model for 14 days were investigated. The five groups included (1) rat allogenic aorta (RAA); (2) decellularized RAA (DRAA); (3) DRAA with S1P (DRAA/S1P); (4) DRAA with EC recellularization (DRAA/EC); and (5) DRAA with S1P and EC recellularization (DRAA/EC/S1P). The result elicited that RAA and DRAA/EC/S1P both had 100% patency without thrombus formation within 14 days. Better endothelialization, more wall structure maintenance and less inflammation were noted in the DRAA/EC/S1P group. In contrast, there was thrombus formation in the DRAA, DRAA/S1P and DRAA/EC groups. Therefore, S1P could inhibit thrombus formation to improve the patency rate of EC-covered decellularized vascular grafts in vivo. In conclusion, S1P is an effective agent capable of decreasing thrombotic risk in engineered blood vessel grafts ex vivo and in vivo and may play an important role in the construction of TEVGs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:10:13Z (GMT). No. of bitstreams: 1 ntu-108-D03b21004-1.pdf: 7422764 bytes, checksum: 2b28122509f5d1aaac8d182214d69eea (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌 謝 ii Abstract vi 目錄 viii List of Tables xii List of Figures xiii Chapter 1 Introduction 1 1.1 Clinical importance of Tissue-engineered blood vessels 1 1.2 Blood vessel scaffolds 2 1.3 Cells utilized for tissue-engineered blood vessels 11 1.4 Blood vessels combined with cells and scaffolds 21 1.5 Biological characters of S1P 24 1.6 Animal model for Decellularizd and recellularized grafts 26 1.6.1 Animal Models for Decellularizd 26 1.6.2 Decellularized Vascular Grafts with Recellularization 28 1.7 Rational 30 Chapter 2 Materials and methods 32 2.1 Ethics assurance 32 2.2 Preparation of DHUV 33 2.3 Isolation, culture, characterization of HUVEC and EPC 33 2.4 Cell staining, seeding on scaffolds, and counting 35 2.5 Coagulation and kinetic clotting tests 36 2.6 Platelet adhesion test 37 2.7 Manipulation of SDC1 expression in recellularized vessels 38 2.7.1 Effect of S1P on SDC1 expression 38 2.7.2 The reaction of MMP-7 on SDC1 39 2.7.3 Platelet adhesion to EC treated with S1P and/or MMP7 40 2.7.4 Statistical analysis 41 2.8 In vivo experiment 41 2.8.1 Preparation and Storage of Native and Decellularized Rat Aortas 42 2.8.2 Primary Culture, Maintenance and Characterization of Rat EC 43 2.8.3 Proliferation Effect of S1P on Rat ECs 44 2.8.4 Effect of S1P on SDC1 Expression in Rat ECs 46 2.8.5 Construction of TEVGs 46 2.8.6 Rat Abdominal Aorta Interposition Graft Model 48 2.8.7 Statistical Analysis 50 Chapter 3. Results 52 3.1 Ex vivo experiments (Figure 1) 52 3.1.1 Decellularization of umbilical vein 52 3.1.2 Characterization of EPCs and HUVECs 52 3.1.3 S1P promotes cell attachment to decellularized DHUV 53 3.1.4 Cellular characteristics of the recellularized DHUV 53 3.1.5 Recellularization in the presence of S1P reduces thrombogenesis in DHUV 54 3.2 In vitro experiments (Figure 1) 56 3.2.1 The effect of S1P on the glycocalyx expression in EC and EPC 56 3.2.2 S1P protects SDC1 on EC and further prevents platelet adhesion 56 3.3 Animal experiments (Figure 14) 58 3.3.1 Characteristics of DRAA 58 3.3.2 Characteristics of Rat ECs 58 3.3.3. Proliferation Effect of S1P on Rat ECs on DRAA 59 3.3.4. S1P Promotes SDC-1 Expression on Rat ECs 60 3.3.5. TEVG Implantation 61 3.3.5.1 Patency Rate and Animal Survival 61 3.3.5.2 Histomorphology of Explanted Vessels 62 3.3.5.3. Endothelialization of Implanted Vessels 63 3.3.5.4. Macrophage Infiltration of Implanted Vessels 64 Chapter 4 Discussion 65 Chapter 5 Conclusions 73 Reference 75 Tables 92 Figures and Legends 100 Appendix I: Abbreviations 123 Appendix II: List of publication 126 | |
dc.language.iso | en | |
dc.title | 鞘氨醇-1-磷酸在組織工程血管構建中之功效及作用機制 | zh_TW |
dc.title | The function and mechanism of Sphingosine-1-Phosphate on endothelial cells during tissue-engineered blood vessel construction | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陸振翮(Jen-Her Lu),姚少凌(Chao-Ling Yao),蔡素宜(Su-Yi Tsai),邱宗傑(Tzeon-jye Chiou) | |
dc.subject.keyword | 鞘氨醇-1-磷酸,凝血,血管內皮細胞,血管內皮前驅細胞,醣蛋白Syndecan-1,組織工程血管, | zh_TW |
dc.subject.keyword | Sphingosine-1-phosphate,Endothelial cell,Endothelial Progenitor,thrombosis,Syndecan-1,TEVGs, | en |
dc.relation.page | 126 | |
dc.identifier.doi | 10.6342/NTU201903087 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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