臺灣博碩士論文加值系統

牙周病的產生會破壞牙周組織的結構，減少牙齒周圍的支撐結構，最後導致牙齒的脫落，牙周病的治療從控制發炎及感染，近來擴展至再生治療的應用，以期恢復牙齒周遭的健康牙周組織。組織工程的進步，發展出許多生長因子的應用。TGF-β及CTGF，在人體或動物體中具有誘導骨再生的能力，。然而在不同組織具有相當的差異性。
研究目的：本研究探討在牙周韌帶細胞中，TGF-β及CTGF對牙周細胞骨再生能力的影響，了解這些生長因子對牙周組織的影響。並且探討TGF-β誘導CTGF訊息傳遞路徑。
材料與方法：本實驗利用牙周韌帶細胞，以TGF-β及CTGF去誘導細胞的骨分化，並檢測其基因表現及鹼性磷酸酶的活性。同時以TGF-β刺激牙周細胞，以西方墨點法檢測 CTGF的訊息傳遞路徑。
結果： TGF-β及CTGF會誘導牙周細胞骨分化基因 BMP2的表現。另外，TGF-β在牙周韌帶細胞中，會經由 JNK、Smad 3、Rac-1、及Src-family kinase 去誘導 CTGF的表現。
結論：TGF-β及CTGF會誘導牙周細胞骨分化基因 BMP2的表現，TGF-β同時會刺激CTGF的表現。因此，TGF-β可能會經由CTGF表現的路徑，去促進細胞的分化，誘導牙周組織的骨分化表現。

關鍵字：牙周再生，轉型生長因子β，結締組織生長因子

Periodontal disease would led to destruction of periodontal tissue, reduce periodontal supporting structure, and finally led to tooth loss. The development of periodontal therapy starts from infection control to tissue regeneration in order to regenerate the healthy tissue surrounding tooth structure. Discovery from tissue engineering found many growth factors leading to tissue regeneration. Recently, transforming growthβ(TGF-β)and connective tissue growth factor (CTGF) can induce bone formation and osteoblast differentiation. However, previous studies show large variety among different cells and tissues
Objectivs: This study is design to find the influence of TGF-β and CTGF on the osteogenic differentiation of periodontal ligament cell,to understand the influence of these growth factors on periodontal tissue. Furthermore, we discover the signal transduction between TGF-β and CTGF to find the mechanism between two growth factors. Therefore, we want to realizing these mechanism and to apply these growth factors to clinical periodontal regeneration
Methods: Immortalized periodontal ligament cells (iPDL) received from Dr. Change of National Yang-Ming University. TGF-β and CTGF were applied to induce the osteogenic differentiation of iPDL. Gene expression was analyzed by Real-time PCR and the protein of differentiation was examined by alkaline phosphatase stain. Further, TGF-β was added to induced CTGF expression in iPDL. CTGF, phosphorylated Smad3, and phosphorlated JNK protein expression were analyzed by western blotting.
Results: TGF-β and CTGF can induce the osteogenic differentiation gene expression in iPDL. TGF-βalso can induce the alkaline phosphatase activity. Furthermore, in PDL, TGF-β can induce the expression of CTGF through JNK, Smad 3, Rac-1, and Src-family kinase.
Conclusion: TGF-β and CTGF can induce osteogenic differentiation in periodntal tissue. TGF-β also can induce CTGF expression in iPDL. Therefore, TGF-β might induce PDL osteogenic differentiation through CTGF expression.

Key words: periodontal regeneration, TGF-β, CTGF

目錄

誌謝 I
中文摘要 II
Abstract III
目錄 V
圖目錄 VIII
第一章 緒論 1
第一節 牙周組織及其細胞 1
1-1 牙周組織 (Periodontium) 1
1-2 牙周韌帶細胞 (Periodontal ligament cell) 1
第二節 骨生理 (Bone physiology) 2
2-1 骨生成及分化過程 2
2-2 鹼性磷酸酶 (Alkaline phosphatase)與骨分化 3
2-3 RunX2與骨分化 4
2-4 成骨蛋白2 (BMP-2)與骨分化 4
第三節 轉型生長因子β (Transforming growth factor-β，TGF-β) 5
3-1 轉型生長因子β介紹 5
3-2 轉型生長因子β的訊息傳遞 6
3-3 轉型生長因子β與骨分化 7
第四節 結締組織生長因子（connective tissue growth factor，CTGF） 8
4-1 結締組織生長因子介紹 8
第二章 研究目的 10
第三章 實驗材料與方法 11
一、細胞株與細胞培養 11
二、藥物處理 12
2-1 轉型生長因子β與結締組織生長因子 12
2-2 Inhibitor使用資料 12
三、誘導牙周韌帶細胞分化 12
四、反轉錄聚合酶連鎖反應 (Reverse Transcription polymerase Chain Reaction；RT-PCR) 13
4-1 RNA萃取 13
4-2 RNA定量 14
4-3 反轉錄 (Reverse transcription；RT) 14
4-4 聚合酵素連鎖反應 (Polymerase Chain Reaction；PCR) 14
4-5 瓊脂凝膠電泳分析 (Agarose gel electrophoresis) 15
五、定量聚合酶連鎖反應 (Real-time Polymerase Chain Reaction；Real-time PCR；qPCR) 15
六、西方墨點法 (Western blot) 16
6-1 蛋白質萃取 16
6-2 蛋白質濃度測定 (Total protein analysis) 16
6-3 膠體配置與電泳分析 17
6-4 蛋白質轉漬 (Protein transfer) 17
6-5 抗體使用以及顯影 18
七、鹼性磷酸酶染色 (Alkaline phosphatase staining) 18
7-1 藥品配置 19
7-2 染色步驟 19
第四章 結果 20
一、不朽化牙周韌帶細胞分化能力 20
二、TGF-β誘導牙周韌帶細胞分化 20
1. TGF-β誘導牙周韌帶細胞分化的基因表現 20
2. TGF-β誘導化牙周韌帶細胞分化的鹼性磷酸酶表現 21
三、CTGF 誘導牙周韌帶細胞分化 22
1. CTGF誘導牙周韌帶細胞分化的基因表現 22
四、TGF-β刺激牙周韌帶細胞 CTGF蛋白的表現 22
1. TGF-β在不同濃度下誘導牙周韌帶細胞之 CTGF的表現 22
2. TGF-β在不同時間下誘導牙周韌帶細胞之 CTGF的表現 23
3.不同抑制劑處理隊 TGF-β誘導牙周韌帶細胞 CTGF表現的影響 23
4. 不同抑制劑處理對 TGF-β誘導不朽化牙周韌帶細胞 CTGF表現的影響 24
5. TGF-β於不同時間下誘導牙周韌帶細胞之 JNK磷酸化的表現 24
6. TGF-β於不同時間下誘導化牙周韌帶細胞之 Smad 3 磷酸化的表現 25
7. Rac-1 inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 JNK 磷酸化的表現 25
8.Src-family kinase inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 JNK 磷酸化的表現 26
9.Rac-1 inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 Smad3 磷酸化的表現 26
10.Src-family kinase inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 Smad3 磷酸化的表現 26
11. Rac-1 inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 Smad2 磷酸化的表現 27
12.Src-family kinase inhibitor 不影響 TGF-β於刺激牙周韌帶細胞之 Smad2 磷酸化的表現 27
第五章 討論 28
第六章 結論 31
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