2017-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/665681摘要：細胞外基質（extracellular matrix）除了作為支撐著組織的基材外，也提供了細胞貼附、生長與分化的信號。本計畫研究將製備聚乳酸-烴基乙酸共聚物（poly(lactic-co-glycolic acid), PLGA）電紡絲纖維支架將做為基體的模板結構，為研究細胞產生之細胞外間質對表面地型的影響我們將製備具排列性與不規則性之兩區域靜電紡絲結構。將研究以細胞分泌細胞外基質的技術，開發由內皮細胞 (endothelial cells)與人類脂肪幹細胞(hASCs)共同培養所產生的細胞外基質支架，我們假設由血管組織細胞所衍生之細胞外基質(cell-derived ECM, or cECM)能具備可誘導再次植入之幹細胞生成新生血管組織的特性。而後，人類臍帶靜脈內皮細胞(HUVECs)或人類脂肪幹細胞(hASCs)將接種在PLGA電紡絲上，培養一段時間後，將細胞去除並且溶解PLGA支架，得到依PLGA模板所構建細胞衍生基質支架(cECM)。脂肪幹細胞將重新被接種在cECM，並且誘導成內皮細胞，我們將使用定量PCR和免疫螢光染色來評估分化的程度。我們預期利用血管組織細胞所分泌之細胞外基質支架，可以促進幹細胞分化成內皮或類內皮細胞。 在in vivo部分，我們採用能提供組織反應與觀察血管新生的一種快速，簡單篩選生物材料的方式: 絨毛尿囊膜模型(CAM model)。我們將觀察hASCs與cECMs在CAM model中的的生物可相容性與觀察其血管再生之效果與新生血管分佈。 我們期待在此研究中根據PLGA靜電紡絲模版種植細胞後能產生分泌有結構與方向性，且具血管誘導性之之細胞衍生細胞外基質支架，改善目前用在誘導血管新生材料上的不足，位來可以用於製作人工微血管組織，應用於需要新生血管之組織修復。 <br> Abstract: Extracellular matrix (ECM) acts as the base structure of tissues and provides appropriate microenvironment to support cell adhesion and direct cell behaviors such as proliferation, and differentiation. In vivo, ECM is initially produced by cells and subsequently formed into a three-dimensional (3D) network. Cell-derived ECM fabricated onto 3D scaffolds has recently garnered increased attention with several studies showing promising results. Evidences indicate that cell-derived ECM (cECM) creates a microenvironment useful for supporting tissue regeneration such as cartilage, bone and skin are demonstrated. However, cECM scaffolds has not been widely applied to highly vascularized tissues. The aim of this proposal is to develop cell-derived extracellular matrix scaffolds for vascular tissue engineering. We hypothesize that ECM derived from human umbilical vein endothelial cells (HUVECs) can be fabricated on the patterned electrospun template and the derived cECM will promoted endothelial differentiation of human adipose tissue-derived stem cells (hMSCs). There are there sections in the proposal: we will first fabricate the PLGA electrospun fibrous scaffold to serve as a structural template for HUVECs to deposit the native ECM materials. Two-region electrospun containing both random and aligned fibers will be fabricated for the observation of the effects of topography of the template for HUVECs (as well as co-culture of hASCs). After being cultured for a period of time, the cell-ECM-PLGA constructs will be de-cellularized and then removal of PLGA templates to form cell-derived ECM scaffolds. The HUVEC-derived ECM will then characterized by SEM, FTIR, XPS, and immunofluorescence staining. Subsequently, hASCs will then be seeded on the cell-derived ECM scaffolds and then induced into endothelial cells in vitro. The endothelial induction of hASCs on the cell-derived ECM scaffolds will be evaluation by qPCR and immunofluorescent staining. Finally, we will examine the in vivo tissue response and evaluation of neo-vasculature network of cECM and hASCs-cECM in the chick chorioallantoic membrane (CAM) model. The samples will be implanted in to the CAM and will be evaluated at 1 day and 1 week post-implantation. The implanted samples and the surrounding tissue will be examined grossly and then fixed, embedded in paraffin, then sectioned. Examination will be taken with hematoxylin and eosin (H&E) staining, or inspected by SEM. We expect that the HUVECs –cECM can be routinely synthesized from the eletrospun template; hASCs differentiation toward endothelial lineage in the cECM scaffold will be induced in vitro; and highly vascularized tissue will be detected in the in vivo simulation (CAM assays) in this project.靜電紡絲細胞外間質脂肪幹細胞血管新生組織工程PLGA electrospun，Extracellular matrixadipose derived stem cellsangiogenesistissue engineering