- 學位論文
幾丁聚醣/硫酸軟骨素/麩胺酸複合材料對於骨髓間葉幹細胞分化潛能之效應
Effects of Chitosan/Chondroitin Sulfate/Glutamic Acid Composite Materials on the Differentiation of Bone Marrow Mesenchymal Stem Cells
摘要
適當的幹細胞增殖分化技術及良好的生醫材料在組織工程應用上極為重要。本研究以細胞分化為目標，針對不同生醫材料的效應進行深入研究及探討。本研究使用幾丁聚醣作為主要之生醫材料，並添加硫酸軟骨素及麩胺酸形成各種複合材料以之進行間葉幹細胞之體外培養，並由光學顯微鏡觀察細胞型態以及細胞DNA的定量來探討生醫材料對間葉幹細胞增殖之影響。其次利用即時定量聚合酶反應 (real-time polymerase chain reaction, real-time PCR ) 觀察在不同分化培養基刺激下各基因表現量。Oct-4及Nanog表現量明顯下降，可看出間葉幹細胞已改變其細胞狀態而朝向特定方向分化。針對骨分化成效觀察細胞之骨鈣素 (osteocalcin, OCN) 基因表現並以Alizarin red S染色觀察鈣分泌；脂肪分化成效觀察過氧化體增殖活化受體 (peroxisome proliferator-activator receptor-γ2, PPAR-γ2) 與脂蛋白酯酶 (lipoprotein lipase, LPL) 基因表現，並以Oil red O染色觀察油滴分布；軟骨分化成效則觀察aggrecan和第二型膠原蛋白 (type II collagen, Col2) 基因表現量及以Toluidine blue及Safranin O染色觀察醣胺素分佈，結果各生醫材料均對分化有較佳效果。 由實驗結果整體來看，幾丁聚醣材料在加入硫酸軟骨素及麩胺酸雖然無法增加間葉幹細胞之貼附與增殖能力，但在分化實驗中卻較控制組 (TCPS材料) 組有更為優越的效果，在細胞型態上也形成與生理上類似的細胞團塊。在骨分化成效上以幾丁聚醣為佳，在脂肪與軟骨分化成效上則以96/4/8 (幾丁聚醣/硫酸軟骨素/麩胺酸複合材料)為佳，將來在骨骼及軟骨組織工程上，十分具有應用潛力。
並列摘要
Suitable stem cell sources with good expansion and differentiation technique, as well as excellent biomaterials are important for tissue engineering applications. In this study, we aimed at the development of new biomaterials for the in vitro expansion and differentiation of bone marrow mesenchymal stem cell (BMSCs). Chondroitin sulfate and glutamic acid were used to modify chitosan in order to prepare composite biomaterials. Inverted microscopy and cellular DNA quantification were used to detect the influence of the biomaterials on the proliferation of BMSCs. Real-time PCR was also used to detect the gene expressions in BMSCs. The gene expressions of Oct-4 and Nanog in BMSCs were lower when they were exposed to a certain differentiation stimuli, thus revealing that cells morphology changed and specific differentiation started. We examine the gene expression of osteocalcin (OCN) and use Alizarin red S staining to detect calcium deposit after BMSCs exposed to osteogenic stimulus (OS), the gene expressions of peroxisome proliferator-activator receptor-γ2 (PPAR-γ2), lipoprotein lipase (LPL) and use of Oil red O staining to detect oil secretion when BMSC were exposed to adipogenic stimulus (AS), the gene expression of aggrecan and type II collagen and use of Toluidine blue and Safranin O staining to detect glycosaminoglycan (GAG) when BMSCs were exposed to chondrogenic stimulus (CS) respectively. In summary, although the addition of chondroitin sulfate and glutamic acid to biomaterial did not improve the attachment and proliferation of BMSCs, the chitosan-based composite biomaterials developed in this study had superior effect than tissue culture polystyrene (TCPS) material in cell differentiation. The chitosan biomaterial was suitable for osteogeic differentiation, while chitosan/chondroitin sulfate/glutamic acid composite biomaterial was suitable for adipogenic and chondrogenic differentiation. Therefore, these biomaterials can be good candidates for bone- and cartilage-repairing tissue engineering applications.
參考文獻
延伸閱讀
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