鞘氨醇-1-磷酸在組織工程血管構建中之功效及作用機制

The function and mechanism of Sphingosine-1-Phosphate on endothelial cells during tissue-engineered blood vessel construction

夏凱(Kai Hsia)

指導教授 ： 李心予

國立臺灣大學/生命科學院/生命科學系/博士(2019年)

https://doi.org/10.6342/NTU201903087

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

Sphingosine-1-phosphate ； Endothelial cell ； Endothelial Progenitor ； thrombosis ； Syndecan-1 ； TEVGs

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