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標題: | 人類間葉細胞在生醫材料上的行為調控和基因型表現 Modulation of Cellular Behaviors and Phenotypic Responses of Human Mesenchymal Cells on Biomaterials |
作者: | Yu-Tsang Lee 李裕滄 |
指導教授: | 楊台鴻 |
關鍵字: | 間葉幹細胞,滑囊細胞,軟骨細胞,奈米磷酸鈣,玻尿酸,生長因子,生醫材料,基因表現, mesenchymal stem cells,synovial cells,cartilage cells,nano-scale calcium phosphate,hyaluronic acid,growth factor,biomaterials,phenotype response, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 本研究的目的是探討人類間葉細胞在生醫材料上的行為調控和基因型表現,研究內容分為兩大部分,第一部分是有關骨組織生成的相關研究,第二部分是有關退化性關節炎的研究。所取用的間葉細胞來自於人體骨髓以及結締組織,包括間葉幹細胞、滑囊細胞、軟骨細胞。
第一部分研究重點在骨生成的組織工程。骨骼成份中無機物主要是磷酸鈣,和第二型膠原蛋白共同組成骨組織的細胞外間質。於實驗室用化合沈澱法方式來製成磷酸鈣的微晶體,並混摻天然有機化合物幾丁聚醣來製成複合型膜狀材料,材料表面因磷酸鈣的結晶性不同而形成奈米至微米大小不同的顆粒。取人體骨盆腸骨骨髓在體外培養間葉幹細胞,並將人類間葉幹細胞培養在不同的幾丁聚醣/磷酸鈣複合材料上,檢測細胞的行為調控,包括細胞生長增殖、細胞貼附分佈,以及在骨誘導培養基下,對鹼性磷酸酵素分泌的情形。研究的結果顯示,在幾丁聚醣/磷酸鈣複合膜狀材料的表面,磷酸鈣的結晶性和表面構形,會影響間葉幹細胞的行為,其中奈米結晶磷酸鈣有明顯提高細胞的增殖,微米結晶磷酸鈣有助細胞的骨系分化。研究的結果,說明材料表面磷酸鈣的結晶體,會增加人類間葉幹細胞的生長增殖和骨系分化,所製作材料可應用於骨骼組織工程支架的改良和表面處理。 第二部分研究重點在探討玻尿酸對膝關節退化症治療的機制。取人體退化性膝關節內的滑囊組織和軟骨組織,在實驗室培養滑囊細胞和軟骨細胞,先用無血清的培養基培養細胞二十四小時,以排除血清內生長因子的作用,選用不同分子量和濃度的玻尿酸,用來刺激細胞四小時及二十四小時,觀察細胞的形態和生存能力的情形,並檢測細胞對退化性關節炎相關生長因子基因型表現的反應,包括結締組織生長因子、轉型生長因子乙型之一、血管內皮生長因子,同時檢測細胞對第一型膠原蛋白和第二型膠原蛋白的基因型表現反應。研究的結果顯示,不同分子量玻尿酸在不同濃度下,對人類滑囊細胞和軟骨細胞作刺激二十四小時,不改變細胞形態,也不會對細胞產生毒性作用,對生長因子的基因型表現有不同的影響,以較高濃度的玻尿酸刺激細胞,會增加生長因子的基因型表現,在同一濃度之下,較高分子量玻尿酸對滑囊細胞作用時,結締組織生長因子和血管內皮生長因子的基因型表現會有下降。研究的結果,對以玻尿酸來治療退化性膝關節炎的機制有進一步的了解,也有助玻尿酸在生醫材料的應用。 Osteogenesis and Osteoarthritis are two big issues in orthopaedic diseases. The main purpose for this study is to understand how to modulate cellular behaviors and phenotypic responses of human mesenchymal cells on different biomaterials. The cells are isolated primarily from human bone marrow and connective tissues, including synovium and cartilage of knee joints. Human mesenchymal stem cells (hMSCs) have great potential to differentiate to lineages of mesenchymal tissues. Calcium phosphate (CaP) apatite, the main inorganic constituent of mammalian bone tissues, is believed to support hMSCs growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the first part, we have applied a simple and quick method to prepare micro- and nano-scale of calcium phosphate (CaP) crystals mixed in chitosan membranes. The different concentrations of aqueous CaP suspension were mixed with chitosan in acetic acid solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. The hMSCs behaviors including cell spreading, proliferation and osteogenic differentiation were examined. In basal culture medium, the addition of CaP in chitosan films could promote the proliferation of hMSCs. The films with nano-crystalline CaP significantly improved cell proliferation. In osteogenic medium, the increased alkaline phosphatase (ALP) level showed the process of osteogenic differentiation of hMSCs on the C/CaP films. The hMSCs on discrete micro-crystalline CaP films revealed higher ALP level. These results demonstrate that the crystallinity and topography of CaP apatite on chitosan membrane scaffolds modulates the behaviors of hMSCs. Intra-articular injection of hyaluronan (hyaluronic acid; HA) is a common way to treat knee osteoarthritis (OA). This treatment can not only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is still unknown. In the second part, human synovial cells were stimulated with HA (Sigma) and Hylan (Synvisc) for 24 hours. The human synovial cells were isolated from synovium tissue of advanced-staged osteoarthritic knee. Real-time polymerase chain reaction (real-time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. The gene expressions of matrix-related proteins, collagen I and collagen II, were also studied. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis in serum deprivation environment. For synovial cells, the cell shapes were not changed after HA and Hylan stimulation for 24 hours. The gene expressions of TGF-β1 and VEGF were significantly increased at the concentration of 0.1mg/ml HA and 0.1mg/ml Hylan, respectively. The synovial cells with treatment of 0.1mg/ml Hylan decreased the CTGF gene expression (0.66-fold) and VEGF (0.78-fold) compared to 0.1mg/ml HA. The type I collagen expressed significantly higher as treated with 0.1mg/ml HA and 0.1mg/ml Hylan. We further isolated human cartilage cells from healthy cartilage of knee. The cartilage cells were treated with three kinds of HA, including Sigma HA, Synvisc Hylan and Artz HA, with 0.1 mg/ml and 0.01mg/ml under serum deprivation condition for 24 hours. With treatment of Synvisc Hylan, the gene expressions of CTGF, TGF-β1,VEGF, collagne I and collage II increased in 0.1mg/ml compared with 0.01mg/ml. However, the gene expression of CTGF, TGF-β1, VEGF, collagne I and collage II decreased in 0.1mg/ml relative to 0.01mg/ml with the treatment of Artz HA. Under the condition of 0.1mg/ml, Artz HA decreased the gene expressions of CTGF(0.8-fold), TGF-β1(0.8-fold) and VEGF(0.5-fold) as compared to Synvisc Hylan. Synvsic Hylan increased the gene expressions of collagen I (1.7-fold) and collagen II (4.9-fold) as compared to Artz HA. As a result, the profile of osteoarthritis-related factors of CTGF, TGF-β1 and VEGF and matrix proteins of collagen I and II might provide the rational mechanism for the therapeutic effects of hyaluronic acid on OA knees. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45933 |
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顯示於系所單位: | 醫學工程學研究所 |
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