臺灣博碩士論文加值系統

牙周病為常見的口腔疾病，也是成年人喪失牙齒的主因。牙周治療的終極目標是希望能再生因牙周病所破壞的組織，但如何確保達成牙周再生仍為現今的一大挑戰。牙周韌帶幹細胞參與牙周組織的癒合，在不同動物模型中已顯示使用牙周韌帶幹細胞有良好促進牙周再生的療效。因為由牙周韌帶組織可分離出的牙周韌帶幹細胞數量十分有限，常須藉長期細胞培養以獲取足夠細胞量，但幹細胞經過長期培養後會有漸漸老化的問題。近期研究顯示採用低氧及低密度方式培養骨髓幹細胞可有效擴增細胞並減緩細胞老化。本研究目的在評估是否同樣的策略可應用於擴增牙周韌帶幹細胞。研究使用分離自五位患者的牙齒並加以等量混合之牙周韌帶幹細胞，將細胞分別培養在常氧或低氧環境(1%氧濃度)以比較細胞表型的改變。常氧組及低氧組細胞在第一代時均高度表達間葉幹細胞標記(CD90及CD105)，而其幹性標記(NANOG及Oct4)的表現也相近。此外，兩組細胞均可分化成骨母細胞及脂肪細胞。比較不同代數細胞的結果，發現相較於低氧培養組，長期培養在常氧環境會導致細胞增殖變慢，倍增所需時間增長，細胞變大變寬，老化標記(SMP-30、p21、Apo1及SA-β-gal)改變增加。進一步以茜素紅染色測試長期培養後的細胞，發現相較於常氧培養，在低氧培養下的細胞較可維持其骨形成潛能，然而以oil red O染色測試，顯示長期常氧及低氧培養均會降低細胞的脂肪形成能力。評估常氧組及低氧組晚繼代數細胞在體內組織再生能力的實驗顯示長期低氧培養下的細胞具有形成似牙周韌帶組織及似牙骨質的能力，及具有分化成夏培氏纖維及造牙骨質細胞的能力，而長期常氧培養的細胞較不易形成牙周相關組織。總括而言，低氧培養有助於減緩細胞老化並維持細胞的體外骨形成能力和體內牙周組織再生能力，有潛力可被應用於大量擴增牙周韌帶幹細胞。本研究的結果將有助於未來應用牙周韌帶幹細胞於牙周再生性治療。

Periodontitis is a common oral disease and a leading cause of tooth loss in adults. The ultimate goal of periodontal therapy is the regeneration of the tissues destroyed as a result of periodontal disease. Achieving predictable periodontal regeneration remains a challenge nowadays. Periodontal ligament stem cells (PDLSCs) are involved in the periodontal wound healing. PDLSC-based therapeutics demonstrates promising results in periodontal regeneration in various animal models. Since PDLSCs isolated from periodontal ligament tissue are quite limited, long-term cell expansion is often mandatory in order to obtain sufficient cell number. Unfortunately, stem cells undergo senescence after extended cell culture. Recent studies indicated that expansion of bone marrow stem cells in a hypoxic and low density manner can facilitate cell expansion and significantly delay the cell senescence. The purpose of this study was to evaluate whether the same strategy can be applied in the expansion of PDLSCs. Pooled periodontal ligament stem cells were isolated from extracted teeth from five subjects and studied. PDLSCs were cultured either under normoxic or hypoxic condition (1% O2) and compared for their changes in phenotypes. At passage one, both cells highly expressed mesenchymal stem cell markers (CD90 and CD105) with comparable expression level of stemness markers (NANOG and Oct4). In addition, both cells could differentiate into osteoblasts and adipoctyes. Results of normoxic and hypoxic PDLSCS at different passages indicated that the long-term normoxic culture, when compared to the hypoxic culture, was accompanied with slower proliferation, increased doubling time, increased cell size and broader morphology, more significant senescence marker changes (SMP-30, p21, Apo1 and SA-β-gal). Furthermore, compared to the normoxic culture, the hypoxic culture can effectively retain the osteogenic potential of cells after long term culture, as evidenced by the results of alizarin red S staining. However, oil red O staining indicated that both normoxic and hypoxic groups showed reduced adipogenic potential at late passage. The regeneration potential of late passage PDLSCs in vivo of hypoxic groups could form cementum-like tissue and PDL tissue, and differentiate into cementoblast-like cells and Sharpey’s fiber. In contrast, cells of normoxic groups had less periodontal regenerative potential. Taken together, the application of hypoxic culture may be beneficial for scale up expansion of PDLSCs through delay in senescence, preservation of osteogenic potential in vitro and periodontal regenerative potential in vivo. The results of this study may facilitate the future use of PDLSCs in periodontal regenerative therapies.

目錄 I
中文摘要 III
英文摘要 V
壹、 緒論 1
一、 研究動機 1
二、 牙周組織 3
三、 牙周病 3
四、 人類牙周韌帶幹細胞 (human periodontal ligament stem cell, hPDLSC) 5
五、 以牙周韌帶幹細胞治療牙周炎之動物研究 6
六、 培養牙周韌帶幹細胞所面臨的問題 7
七、 低氧環境及低密度繼代培養對幹細胞的影響及重要性 7
貳、 實驗材料與方法 11
一、 實驗材料 11
壹、 細胞培養液 11
貳、 生化分生試劑、套組 12
參、 動物實驗 16
肆、 儀器 17
二、 實驗方法 18
1. 分離人類牙周韌帶幹細胞 18
2. 混合五位患者之牙周韌帶幹細胞及低密度細胞培養 18
3. 細胞培養 19
4. 流式細胞儀實驗步驟 20
5. 人類牙周韌帶幹細胞及人類骨髓間葉幹細胞分化實驗 20
6. 茜素紅(Alizarin red S)染色法 21
7. 油紅O (Oil red O)染色法 21
8. 測試老化實驗(Senescence-associated beta-galactosidase activity) 22
9. 西方點墨法 23
10. 動物實驗 25
11. 蘇木紫-伊紅染色法(hematoxylin-eosin stain, H&E stain) 26
12. 統計方法 28
參、 實驗結果 29
一、 分離人類牙周韌帶幹細胞 29
二、 確認牙周韌帶幹細胞的表徵 29
三、 測試分離出來的牙周韌帶幹細胞其分化潛能 30
四、 比較常氧組及低氧組細胞的增殖速率 31
五、 比較不同代數細胞的形態 32
六、 比較常氧組及低氧組細胞老化酵素senescence-associated β-galactosidase (SA-β-gal)的表現 32
七、 比較常氧組及低氧組細胞衰老標記蛋白senescence marker protein-30 (SMP-30)的表現 33
八、 比較常氧組及低氧組細胞衰老標記p21及Apo1的表現 33
九、 比較常氧組及低氧組不同代數細胞骨分化的能力 34
十、 比較常氧組及低氧組不同代數細胞脂肪分化的能力 35
十一、 體內測試常氧及低氧細胞再生的能力 36
肆、 討論 38
伍、 結論 44
陸、 附圖 45
圖一、人類牙周韌帶幹細胞的形態 45
圖二、利用流式細胞儀確認牙周韌帶幹細胞的表徵 46
圖三、利用QPCR測量牙周韌帶幹細胞的幹性表現 47
圖四、分離的牙周韌帶幹細胞具骨分化及脂肪分化能力 48
圖五、比較常氧組及低氧組細胞的增殖速率 49
圖六、比較不同代數細胞的形態 50
圖七、比較P5常氧組及低氧組細胞老化酵素SA-β-gal的表現 51
圖八、比較常氧組及低氧組細胞衰老標記蛋白Senescence marker protein-30 (SMP-30)的表現 52
圖九、比較常氧組及低氧組細胞衰老標記p21及Apo1的表現 53
圖十、比較常氧組及低氧組早期代數細胞骨分化的能力 54
圖十一、比較常氧組及低氧組早期代數細胞OCN的表現量 55
圖十二、比較常氧組及低氧組晚期代數細胞骨分化的能力 57
圖十三、比較常氧組及低氧組晚期代數細胞OCN的表現量 58
圖十四、比較常氧組及低氧組早期與晚期代數細胞骨分化的能力 59
圖十五、比較常氧組及低氧組早期代數細胞脂肪分化的能力 60
圖十六、比較常氧組及低氧組晚期代數細胞脂肪分化的能力 61
圖十七、比較常氧組及低氧組早期與晚期代數細胞脂肪分化的能力 62
圖十八、動物實驗流程圖 63
圖十九、比較常氧組及低氧組細胞體內形成類似牙周組織結構的能力 65
圖二十、植入體內八週後蘇木紫-伊紅染色觀察膠原蛋白纖維 66
柒、 參考文獻 67

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