臺灣博碩士論文加值系統

本研究在投藥促使人類牙髓幹細胞 (Human dental pulp stem cells,DPSC )往成骨細胞方向分化的條件下，同時以直流電進行直接電刺激，以探討電刺激在幹細胞骨分化過程中的影響。首先 DPSC 在可
導電之聚吡咯薄膜 Polypyrrole (PPy) 上進行培養，待細胞貼附後更換培養液為誘導骨分化培養基，並對電刺激組於第 0 天施加單次直流
電刺激，在第 14 天以鈣含量和細胞數的測定分析細胞分化能力。結
果顯示通電後細胞的分化能力增加至 2 到 3 倍，得知直接電刺激可顯
著增加 DPSC 的骨分化能力。接著利用定量即時聚合酶鏈鎖反應
(quantitative real-time PCR) 在第 0 天通電之後分別於第 0﹑2﹑4﹑6﹑8 和 10 天對電刺激組和非電刺激組別進行和骨分化相關基因的相對
表現量測定，發現 BMPs、VEGF、Runx-2 和 CALM 在培養前期有正向上調控，在培養後期 ALP、Col-1、Osx 和 BGLAP 受電刺激影響增加表現。而後由西方墨點法分析磷酸化 SMAD 表現結果證實：無論是否在有骨分化培養基的情況下，直接電刺激都明顯使 SMAD 磷酸化的比例增加，推測直接電刺激是藉由影響 SMAD 的訊息傳遞路徑影響幹細胞骨分化的能力。另外為找出較佳的電刺激時間點，規劃在不同時間點施加一次性的直接性電刺激，在第 0﹑2﹑4﹑6﹑8 和第10 天分別施加電刺激，各自在第 12﹑14 和第 21 天進行礦化程度的分析，得知於第 4 天通電的組別在促進骨分化的表現特別顯著。從另一個角度來看，從第 14 天之後的礦化分析結果得知，電刺激組增加礦化效果達到高，爾後隨培養時間拉長增加效果逐漸減少，因此判斷直接電刺激的輔助效果為加速骨分化而非增加骨生成的質量。

Substrate-mediated direct current (DC) was studied the effects of electrical stimulation (ES) on human dental pulp stem cells (hDPSC) during osteogenic process. Conductive polypyrrole (PPy) films were prepared as substrate for cell seeding. Osteogenic medium was applied to trigger cell differentiation, and a constant electric field (2 V/6 cm) was applied for 4 h once medium was supplied osteogenic contents. At Day 14, the mineralization of hDPSCs treated with DC stimulation were
highly improved that its calcium deposition was 2 to 3 times more than the untreated group. The transcriptions of osteo-differentiation relative genes were examined using quantitative real-time polymerase chain reaction (qPCR) at different days. The DC treatment was capable of immediately leading the enhancement of the transcriptions of BMPs, VEGF, Runx-2, and Osx, by which ALP, Col-1, and BGLAP were thus up-regulated with time. Although the levels of SMAD-1/5 were not affected, the Western blot results indicated that the phosphorylations of SMAD-1/5 were highly improved. It suggested the ES likely activated SMAD signal pathway to upregulate Runx-2 then improved osteogenesis. Finally, hDPSCs at different stages of osteo-differentiation were individually treated ES. The mineralization results revealed that osteogenesis were enhanced when hDPSCs were treated ES at early stages of osteo-differentiation, especially at Day 4. In contrast, there were almost no differences of mineralizations between the ES treatments at late stages and the untreated group. The improvement was extremely obvious for the calcium deposition at Day 12. However, the saturated calcium deposition of ES treated and untreated groups at Day 21 were almost the same. These results suggested that ES treatment likely accelerated osteo-differentiation.

摘要 I
Abstract III
致謝 IV
目錄 VI
圖表目錄 VIII
第一章 緒論 1
1-1 研究動機 1
1-2 實驗目的 4
第二章 文獻回顧與理論基礎 5
2-1 組織工程 5
2-2 幹細胞 7
2-3 牙髓間質幹細胞 9
2-4 骨分化過程簡述 11
2-5 骨分化相關生物分子及基因 13
2-6 電刺激 19
第三章 實驗方法與設備 23
3-1實驗藥品及儀器 23
3-2 試劑材料製備與實驗方法 29
3-2-1 Polypyrrole製備、電刺激裝置製作 29
3-2-2細胞冷凍解凍、培養繼代 31
3-2-3 電刺激與骨分化條件 33
3-2-4 MTT Assay 36
3-2-5 LDH Assay 37
3-2-6茜素紅染色(Alizarin Red Staining, ARS) 38
3-2-7 Calcium-O-Cresophtalein Complexone 39
3-2-8 Purification of Total RNA from Animal Cells Using Spin Technology-RNeasy Mini Kit 41
3-2-9 SuperScript III First-Strand Synthesis System for RT-PCR 43
3-2-10 Real-Time PCR (Quantitative Polymerase Chain Reaction) 45
3-2-11 蛋白質定量分析 46
3-2-12 蛋白質體電泳 (SDS-PAGE) 47
3-2-13西方墨點分析法 (Western Blot analysis) 50
3-3 實驗架構設計 54
3-3-1 直接電刺激對骨分化的影響 54
3-3-2 不同時間點電刺激對骨分化的影響 55
3-4 生物性質分析 56
3-4-1 茜素紅染色(Alizarin Red Staining, ARS) 56
3-4-2 Calcium-O-Cresolphthalein Complexone 57
3-4-3 即時聚合脢連鎖反應real-time PCR 58
3-4-4 西方墨點法 60
第四章 結果與討論 61
4-1 直接電刺激對骨分化的影響 61
4-1-1 茜素紅染色與鈣離子沉積分析骨分化效果 61
4-1-2 即時聚合脢連鎖反應 ( Real-Time PCR ) 65
4-1-3 西方墨點法 79
4-2 不同時間點電刺激對骨分化的影響 81
4-2-1 茜素紅與鈣離子沉積分析骨分化效果 81
第五章 結論 90
參考資料 92

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