- 學位論文
人體血小板裂解液對於脂肪幹細胞薄片增生及其促進傷口修復潛力之探討
The effects of platelet lysate on adipose-derived stem cell sheet formation and its potential wound healing capability
摘要
細胞層片已被應用於再生醫學領域多項組織工程及疾病治療，而脂肪幹細胞為近幾年相當具有潛力的細胞治療選項。諸多研究將脂肪幹細胞以細胞層片的形式應用在組織再生上已獲得顯著的實驗成果。然而傳統的細胞培養需使用動物來源血清如胎牛血清，此類血清有傳播病原體即引發臨床併發症的危險，且有動物福祉的考量。與此相比，人體血小板裂解液提供了我們一個相當具有吸引力的血清替代物。在本研究中，我們使用人體血小板裂解液來替代胎牛血清，並比較兩種血清所培養出來的脂肪幹細胞層片有何差異性。 在本研究中，脂肪幹細胞是以基底培養液加入胎牛血清或人體血小板裂解液後再加入A-2P所培養。A-2P為維他命C一衍生物，在其刺激下，細胞會大量分泌細胞外基質進而形成一層狀結構。在兩種血清的培養下，使用人體血小板裂解液所培養的脂肪幹細胞具有更快速的生長速度及更高的骨分化能力，然而其脂分化能力相較於使用胎牛血清培養的脂肪幹細胞層片則是有所下降。我們進一步的比較了兩種血清所培養的細胞層片其基因及蛋白表現，以及測試了從兩種層片所收集來的培養液對纖維母細胞及血管內皮細胞的影響。其中來自人類血小板裂解液所培養脂肪幹細胞層片的培養液有效的促進了纖維母細胞及血管內皮細胞的增生及爬行，這些結果有效的指出人類血小板裂解液所培養出的脂肪幹細胞層片在促進傷口癒合上相較使用胎牛血清所培養出的脂肪幹細胞層片來的更有潛力。雖然當我們將兩種血清所培養的脂肪幹細胞層片植入雞胚尿囊膜時組別間血管生長的情況並沒有顯著趨勢，但未來仍可望使用其他更穩定的模型來驗證人體血小板裂解液所培養之脂肪幹細胞層片在促進傷口修復上的可能性。
並列摘要
Adipose-derived stem cells (ASCs) holds a valuable future in regenerative medicine. ASC sheet has been suggested to promote tissue healing and has been applied as possible treatment in a variety of disease models. However, the use of animal-derived serum supplemented growth media may lead to concerns regarding to clinical complication. In recent years, human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for the ex-vivo expansion of mesenchymal stem cells for clinical use. In this study, we compare ASC sheets that have been cultured in growth media supplemented with either FBS or HPL. ASCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose supplemented with either FBS or HPL. Cell sheet formation was further induced by the addition of ascorbic acid 2-phosphate. We performed quantitative PCR, western blotting, ELISA and proteomic analysis to further compare ASC sheets culture with FBS and HPL. In this study, we found that ASC sheets cultured with HPL acquired a higher population doubling and better osteogenic differentiation, though adipogenic differentiation was reduced relative to ASC sheet cultured with FBS. Proteomic analysis of sheet extracellular matrix showed a much more abundant extracellular matrix deposition. ASC sheets cultured in HPL-supplemented growth medium exhibit enhancing effects on the proliferation and migration of dermal fibroblasts and endothelial cell. Conditioned medium collected from ASC sheets cultured in with HPL showed significantly higher proliferation and migration rates when used to cultured fibroblasts cell line HS68. It also results in more tube formation in the in-vitro HUVEC angiogenesis assay when compared to medium collected from ASC sheets cultured with FBS. Further analysis revealed that HPL-supplemented culture medium significantly enhanced expression of hepatocyte growth factor (HGF) and chemokine (C-C motif) ligand 5, and reduced expression of IL-6 in ASC sheets, which have all been shown to play important roles in angiogenesis and wound healing process. Overall, Supplementation of HPL promoted a faster and more robust ASC sheet formation, and the secretion of angiogenic paracrine factors from ASC sheets was also enhanced. ASC sheet cultured with HPL has been shown to promote in vitro fibroblasts and endothelial cells proliferation and migration. Though the mechanism behind these results is yet to be confirmed, these in-vitro test results demonstrated a potential wound healing capability of HPL-cultured ASC sheet in addition to circumventing the untoward side effects for using FBS for cell culture. Therefore, HPL-cultured ASC sheet exhibits great potential in future clinical application to promote better wound healing.