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標題: | 周期性張力合併葡萄糖胺對人類牙根尖乳突細胞成骨分化之影響 Combination Effect of Cyclic Tension Force and Glucosamine on Osteogenic Differentiation of Human Apical Papilla Cells |
作者: | Yi-Tzu Lin 林怡孜 |
指導教授: | 陳羿貞(Yi-Jane Chen) |
共同指導教授: | 鄭景暉(Jiiang-Huei Jeng) |
關鍵字: | 人類牙根尖乳突細胞,成骨分化基因,周期性張力刺激,葡萄糖胺,Cbfa1/Runx2, human apical papilla cells,osteogenic genes,cyclic tensional force,glucosamine,Cbfa1/Runx2, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 骨組織工程的三個要素包括細胞、組織生長支架與生長信息分子,間葉幹細胞在骨組織工程領域中扮演重要的角色,其來源可從胚胎或成熟個體取得。人類牙根尖乳突是指在牙根尚未發育完全之恆牙牙根末端組織,由該組織分離出來的牙根尖乳突細胞具有間葉細胞及幹細胞的特性,在適當的刺激環境中,牙根尖乳突細胞具有與骨髓間葉幹細胞相似的成骨分化能力。回顧文獻中有許多研究證實機械力量刺激會促進骨髓間葉幹細胞的成骨分化,我們實驗室團隊的研究發現機械張力刺激可能也參與牙根間乳突細胞分化的調控;另一方面也證實葡萄糖胺 (glucosamine)小分子會經由提升TGF-β第一型接受器表現而促進牙髓幹細胞 (dental pulp stem cell) 的成骨分化。因此,本研究的目標是探討同時給予物理性 (cyclic tension force) 與化學性 (glucosamine) 二種刺激是否對於牙根尖乳突細胞的成骨分化具有加成效果。本研究所使用的人類牙根尖乳突細胞取自年輕患者之下顎第三大臼齒並以第五代至第八代的細胞來進行實驗。除了觀察人類牙根尖乳突細胞於受力或葡萄醣胺培養下的形態變化之外,並以螢光染色觀察細胞骨架在藥物抑制劑及受力下結構變化,最後分別以即時聚合酶鏈鎖反應、西方墨點法觀察成骨基因及蛋白質在合併周期性張力與葡萄醣胺刺激之表現。本研究結果顯示受張力刺激使人類牙根尖乳突細胞在排列方向上較具有一致性,細胞骨架也較具有方向性且緻密度較高;接受葡萄醣胺作用的細胞在形態上與對照組近似,但是生長速率低於對照組。螢光染色觀察結果顯示Cytochalasin D造成細胞骨架結構的破壞,actin filament的排列變得雜亂無章;Jasplakinolide則使細胞骨架呈現僵化現象,在細胞核周圍出現明顯濃染。以西方墨點法分析Cbfa1/Runx2蛋白質的表現,結果發現Cytochalasin D與Jasplakinolide這兩種藥物都會使Cbfa1/Runx2表現量降低,由實驗結果可知上述兩者因為使細胞骨架失去動態變化能力而影響了成骨分化基因 (Cbfa1/Runx2) 的表現。在成骨分化基因 (Cbfa1/Runx2、ALP、OCN) 及Cbfa1/Runx2蛋白質表現方面,同時給予周期性張力及葡萄醣胺刺激的細胞,其Cbfa1/Runx2、ALP基因表現量在48小時候可見加成效果(additive effect)且達到統計上的顯著性;成牙本質分化的基因DSPP雖然表現量也有增加,但幅度不若成骨基因明顯;Cbfa1/Runx2蛋白質磷酸化表現也具有加成效果,但是程度不若基因表現來得明顯,單獨給一種刺激時,Cbfa1/Runx2磷酸化程度比起對照組都有上升趨勢。過去研究顯示人類牙根尖乳突細胞屬於較原始的幹細胞,如果在適當的誘導後移植於體內環境,牙根尖乳突細胞可表現其成牙本質分化潛能而有助於再生牙根再生技術;更多研究發現牙根尖乳突細胞於體外培養時,其成骨分化能力表現會優於成牙本質分化能力,因此牙根尖乳突細胞可應用於骨再生及組織工程技術。本研究發現同時給予物理性及化學性刺激對於人類牙根尖乳突細胞的成骨分化在基因表現方面具有加成作用;Cbfa1/Runx2蛋白質磷酸化的加成作用雖然沒有基因表現明顯,但上升程度仍具有統計學上的意義。將來的研究方向可著重在晚期成骨基因及蛋白質的表現,以及礦化基質沉積之觀察。 The three key ingredients for tissue engineering are cells, scaffolds, and growth-stimulation signals. Mesenchymal stem cells play an important role in bone tissue engineering. The source of mesenchymal stem cells could be embryonic or somatic origin. Previous studies reported that the progenitor cells obtained from apical papilla tissue of developing teeth exhibit the characteristic of mesenchymal stem cells. Apical papilla stem cells (APSCs) could commit to osteogenic differentiation in vitro as bone marrow mesenchymal stem cells. Our previous work revealed that low-level mechanical tensional force upregulated the osteogenic differentiation of human apical papilla cells. Moreover, the amino sugar glucosamine promoted the osteogenic differentiation of dental pulp stem cells through modulating the level of the TGF-β1 receptor. The purpose of the present study was to evaluate whether glucosamine could act synergistically with cyclic mechanical tensional force to induce osteogenic differentiation of human dental apical papilla cells. Human apical papilla cells (APCs) was obtained from apical tissues of developing third molars in young patient. Cells in passage 5 – 8 were used in the present study. After subjecting to cyclic tension force in the presence or absence of glucosamine, the mRNA expression of osteogenic genes in APCs were examined by using RT-PCR techanique, and the protein expression of Cbfa1 was analyzed by western blotting. The results revealed that the cytoskeleton in stretched APCs was aligned perpendicular to strain axis. The morphology of APCs exposed to glucosamine was not significantly different from the un-treated control cells. The treatment of cytochalasin D disrupted the organization of actin filaments, which became more obvious when cells subjecting to mechanical tensional force. Similarly, the treatment of jasplakinolide resulted in paradoxical stabilization of actin filament, which was not altered by mechanical tension force. Moreover, expression of Cbfa1/Runx2 (Core-binding factor alpha-1/Runt-related transcription factor-2) protein was inhibited by cytochalasin D and jasplakinolide. These findings confirmed that an intact, dynamic cytoskeleton is required for stretch-induced expression of Cbfa1/Runx2 in APCs. The expression of Cbfa1/Runx2 and ALP (alkaline phosphatase) genes were significantly upregulated in the APSCs subjected to cyclic mechanical tensional force and glucosamine treatment for 48 hours. The mRNA expression of DSPP (dentin sialophosphoprotein) gene was also upregulated. Moreover, the transcription factor Cbfa1/Runx2 was activated by either mechanical tensional force or glucosamine treatment, and the combination of both. The additive effect of the physical and chemical stimuli was noted for the phosphorylatiobn of Cbfa1, but not for the expression of Cbfa1 protein. To our knowledge, the present study was the first to report the combination effect of glucosamine and cyclic mechanical tensional force in upregulating the osteogenic differentiation in APSCs. Further study is required to further investigate the long-term effects, e.g. mineralized matrix deposition. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17303 |
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