- 學位論文
人類牙髓幹細胞球體之體外血管新生效應探討
The Angiogenic Effects of Human Dental Pulp Stem Cells Spheroids in Vitro
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摘要
相對於傳統平面培養的模式，多細胞球體培養有利於細胞與細胞、細胞與胞外基質之間的訊息傳遞；球體培養也能造成球體內部缺氧而促成缺氧因子的表現及反應。本研究旨在探討人類牙髓幹細胞球體培養是否具有增進血管新生的特性。 研究材料為取自齒顎矯正病人拔除之智齒培養的初代牙髓幹細胞，將細胞培養於幾丁聚醣薄膜使之聚集形成多細胞球體，對照組為傳統培養的平面細胞。研究方法包括牙髓幹細胞形態分析、細胞活性測試、缺氧及血管新生因子之蛋白質表現與mRNA表現分析。利用西方墨點法分析蛋白質表現，並以反轉錄聚合酶連鎖反應分析上游基因的調控變化。此外，本研究尚收集牙髓幹細胞球體之條件培養基，並利用人類臍靜脈內皮細胞進行體外血管形成試驗，探究細胞分泌物質是否具有促進血管新生的潛能。 研究結果顯示牙髓幹細胞球體形成初期結構較為鬆散；隨著時間推進，球體尺寸增加，細胞之間的排列也更加緊密。在經過多天培養後，將球體細胞分散後再回種培養，發現仍具細胞活性並能持續增殖。蛋白質分析顯示成球培養模式可誘發牙髓幹細胞處於缺氧環境，成球培養二天後的缺氧誘導因子-1α(HIF-1α)蛋白質表現上升，培養三天後差異仍然顯著；血管內皮生長因子-A (VEGF-A)在成球培養二天後也呈現顯著上升；至於血管生成素1 (Angptn-1)的表現在成球培養下僅有些許上升的趨勢。反轉錄聚合酶連鎖反應分析顯示:成球牙髓幹細胞的缺氧誘導因子1α基因的mRNA在第一天即有顯著上升；血管生成素1基因在第三天的mRNA表現顯著上升；血管生成素2的mRNA表現於第一天顯著提高。其他相關血管新生因子之mRNA表現，包括血管內皮生長因子A與鹼性纖維母細胞生長因子，在平面培養與球體培養的牙髓幹細胞表現皆無顯著差別。在體外血管形成試驗方面的結果，培養成球牙髓幹細胞之條件培養基能夠顯著促進人類臍靜脈內皮細胞形成管狀結構，具有促進內皮細胞血管新生的功能。 總結而言，本研究闡明在幾丁聚醣薄膜上培養之球體牙髓幹細胞的血管新生效應優於傳統平面培養細胞，具有應用於傷口修復以及組織再生的潛能。
並列摘要
Three-dimensional (3D) spheroid culture has been recognized to support intercellular communication and mimick native tissue microenvironment in vivo. This study aimed to investigate angiogenic potential of human dental pulp stem cells (DPSCs) cultured as 3D multicellular spheroids. Primary DPSCs were isolated from dental pulp tissues extracted from orthodontic patients' third molars. DPSCs were cultured on chitosan membranes to form multicellular spheroids, while the control cells were cultured as 2D monolayers on conventional tissue culture polystyrene (TCPS) dishes. The comparison between spheroids and 2D monolayer cultures included cell morphology, cell viability, protein expression and mRNA expression of hypoxia- and angiogenesis-related factors. The conditioned media from the cultured DPSCs were collected for tube formation assay on human umbilical vein endothelial cells (HUVECs) to evaluate the angiogenic potential of secreted factors. The results demonstrated that the initially formed DPSC spheroids exhibited a loose structure. As time progressed, the spheroid size increased, and the arrangement of cells became more compact. The size of individual cells of spheroid culture was smaller than that of 2D monolayer culture. After dissociation and reseeding, the cells from spheroids could proliferate steadily with culture time. The spheroid culture induced a hypoxic environment for DPSCs, as evidenced by an upregulation of hypoxia-inducible factor-1α (HIF-1α) protein expression in the 2-day spheroid culture, and the difference was still significant in the 3-day culture. The protein expression of vascular endothelial growth factor-A (VEGF-A) was also enhanced in 2-day spheroid culture, while the angiopoietin-1 (Angptn-1) exhibited only a slight increasing trend. The reverse transcription polymerase chain reaction (RT-PCR) revealed that the mRNA levels of hypoxia-inducible factor-1α and angiopoietin-2 significantly increased in the 1-day spheroid culture, the angiopoietin-1 gene showed significant expression in the 3-day culture. The mRNA expression of other angiogenesis-related factors, including vascular endothelial growth factor-A and basic fibroblast growth factor, showed no significant differences between monolayer and spheroid DPSC cultures. In terms of the results of the tube formation assay, conditioned media from spheroid-cultured DPSCs significantly promoted the formation of tubular structures by human umbilical vein endothelial cells, indicating a pro-angiogenic function in promoting endothelial cell blood vessel formation. In summary, our study demonstrated that the angiogenic potential of DPSCs cultured as spheroids on chitosan membranes was superior to that of conventional monolayer culture, highlighting their potential in the application for wound healing and tissue regeneration.
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