博碩士論文 104324065 詳細資訊



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姓名 徐瑋呈(Wei-Cheng Syu)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 直接電刺激透過鈣離子-攜鈣蛋白依賴性激酶二型活化SMAD蛋白訊號以促進人類牙髓幹細胞骨分化
(Direct Electrical Stimulation Activates SMAD Protein Signaling through Ca2+-Calmodulin-dependent Protein Kinase II to Promote Osteogenesis of Human Dental Pulp Stem Cells)
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摘要(中) 本研究將人類牙髓幹細胞(human dental pulp stem cells, hDPSCs)培養於導電高分子薄膜表面，並施以直流電，以探討電刺激對於細胞內促進骨分化的機轉。首先利用ELISA分析，發現電刺激不會增加BMP-2蛋白的表現。再來透過有綠色螢光的鈣離子追蹤劑 Fluo-4，發現電刺激會提高胞內鈣離子濃度。接著輔以抑制劑處理來確認鈣離子的來源，發現電壓依賴性鈣離子通道的抑制劑Nifedipine可有效地抑制電刺激對胞內鈣離子的提升。利用西方墨點法分析分析攜鈣蛋白激酶二型(CaMKII)與SMAD1/5&SMAD3的蛋白及其磷酸化表現，發現Nifedipine造成鈣離子濃度下降，會下調CaMKII與SMAD1/5&SMAD3蛋白的磷酸化程度。另外，我們使用CaMKII磷酸化的抑制劑KN-62，並發現對SMAD1/5&SMAD3的磷酸化一樣會有下調控的效果，證實鈣離子是間接透過CaMKII影響SMAD1/5&SMAD3的磷酸化而非直接對SMAD1/5&SMAD3造成影響。最後我們利用茜素紅和鈣沉積法分析在抑制劑存在的情況下電刺激是否能促進骨分化。結果證實相較於沒有施加電刺激的控制組，在有電刺激的組別骨基質增加至1.5倍，但是在加入抑制劑Nifedipine或KN-62的情況下電刺激的促進效果均被顯著地抑制。這些結果證實直接電刺激應該是經由電壓依賴性鈣離子通道增加鈣離子，促使CaMKII的磷酸化，導致SMAD1/5&SMAD3磷酸化的上調，進而透過BMP&TGF-β路徑增進調控轉錄因子的表現以促進細胞骨分化。
摘要(英) In this study, human dental pulp stem cells (hDPSCs) were cultured on conductive polymer films which were treated direct current to explore the mechanism of electrical stimulation (ES) on the promotion of osteogenic differentiation. The results of ELISA analysis indicated that the ES did not increase BMP-2 expression. Next, a green-fluorescent tracer, Fluo-4, was applied to trace calcium ions, and the results suggested that ES increased the levels of intracellular calcium. To confirm the source of increasing calcium, different inhibitors were applied during ES treatment. Nifedipine, a voltage-dependent calcium channel (VDCC) inhibitor, effectively inhibited the improvement of intracellular calcium under ES. Moreover, Western blot analysis was applied to analyze the expressions of calmodulin-dependent protein kinase II (CaMKII) and SMAD1/5&SMAD3 protein and their phosphorylation. The results showed that the reduction of Ca2+ caused by Nifedipine down-regulated the phosphorylations of both CaMKII and SMAD1/5&SMAD3. To confirm that the CaMKII phosphorylation was required for the up-regulation of SMAD1/5&SMAD3 phosphorylation, KN-62, an inhibitor which blocks the combination of CaM and CaMKII, was applied to inhibit CaMKII phosphorylation. Its inhibition resulted in the reduction of SMAD1/5&SMAD3 phosphorylation in ES treated cells, suggesting the SMAD1/5&SMAD3 signal pathway activated by ES was mediated by CaMKII phosphorylation. Finally, these inhibitors were applied to determine their effects on mineralization of ES-treated cells, which were evaluated by Alizarin Red staining and Ca-OCPC complex method. The mineralization of ES treated cells was 1.5 times that of the untreated cells when there was no inhibitor. However, this improvement was significantly reduced as the cells were treated Nifedipine or KN62. These results suggesting that ES should increase intracellular Ca2+ via VDCC to promote phosphorylation of CaMKII, which leads to up-regulation of SMAD1/5&SMAD3 phosphorylation and further enhances the expression of transcription factors through the BMP pathway and eventually facilitate cell differentiation.
關鍵字(中) ★ 電刺激 關鍵字(英)
論文目次 致謝III
摘要 I
Abstract II
目錄III
表目錄VIII
圖目錄 IX
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
2.2.1 牙髓間質幹細胞 9
2.3 骨分化過程11
2.3.1 SMAD 蛋白家族 (SMAD protein family) 13
2.4 電刺激15
2.4.1 電刺激對細胞之作用 17
2.4.2 電刺激對細胞骨質生成之作用18
2.5 鈣離子訊號傳導19
2.5.1 細胞內鈣離子之作用 19
2.5.2 鈣離子通道(Calcium Channels)20
2.5.3 鈣訊號傳導與骨分化之關係23
第三章 實驗材料與方法 27
3.1 實驗藥品27
3.1.1 導電材料製備藥品 27
3.1.2 細胞培養、骨分化用藥27
3.1.3 骨分化定性、定量試劑28
3.1.4 BMP-2 ELISA Kit 試劑29
3.1.5 Fluo-4-AM 鈣離子監控試劑29
3.1.6 鈣訊號傳導相關蛋白抑制劑29
3.1.7 蛋白質萃取、定量、定性試劑30
3.1.8 免疫螢光染色試劑 31
3.2 實驗儀器32
3.3 試藥製備與實驗方法33
3.3.1 Polypyrrole film 與電刺激裝置製備33
3.3.2 細胞繼代培養與冷凍、解凍34
3.3.3 骨分化培養液配方 (Osteogenesis medium)36
3.3.4 BMP-2 ELISA 定量分析 37
3.3.5 Fluo-4-AM 鈣離子監控分析39
3.3.6 蛋白質收樣與定量分析40
3.3.7 蛋白質膠體電泳(SDS-PAGE)41
3.3.8 西方墨點分析法 (Western Blot analysis)44
3.3.9 免疫螢光染色法 48
3.3.10 茜素紅染色定性分析 (Alizarin Red Staining) 51
3.3.11 Calcium-O-Cresophtalein Complexone 定量分析52
3.4 實驗架構設計55
3.4.1 電刺激對骨型態發生蛋白的影響57
3.4.2 電刺激對細胞內鈣離子調控作用58
3.4.3 電刺激下鈣調控對蛋白質分子機制的影響59
3.4.4 電刺激下鈣調控對細胞骨分化的影響60
第四章 結果與討論 61
4.1 電刺激對骨型態發生蛋白的影響 61
4.1.1 BMP-2 蛋白定量分析 61
4.2 電刺激對細胞內鈣調控之影響 62
4.2.1 細胞內鈣離子的監控 62
4.2.2 電刺激對鈣調控相關蛋白之影響64
4.3 電刺激下抑制劑對鈣調控之影響 66
4.3.1 電刺激下鈣離子增加之來源66
4.3.2 電刺激下抑制劑對鈣相關蛋白之影響68
4.3.3 鈣調控對 SMAD1/5&SMAD2/3 蛋白之影響70
4.3.4 SMAD1/5&3 蛋白磷酸化之移位 76
4.4 電刺激下鈣調控對細胞骨分化的影響 81
4.4.1 茜素紅染色定性分析 81
4.4.2 鈣離子沉積定量分析 83
第五章 結論 85
參考文獻89
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指導教授 胡威文(Wei-Wen Hu) 審核日期 2017-8-21
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