博碩士論文 103324037 詳細資訊



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姓名 陳昱君(Yu-Jyun Chen)  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 人類牙髓幹細胞在直接電刺激其細胞週期與骨分化之關聯性
(The Relationship between Cell Cycle and Osteogenesis of Human Dental Pulp Stem Cells under Direct Electrical Stimulation)
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摘要(中) 本研究分別在一般培養(culture medium, CM)與投入骨分化藥物培養(osteogenesis medium, OM)下，對人類牙髓幹細胞(Human dental pulp stem cells, hDPSC)以直流電進行單次直接電刺激，探討電刺激對幹細胞在其細胞週期與骨分化之關聯性。鈣沉積量與細胞數分析結果顯示通電後分化能力提升至1.5到2倍且細胞數有下調。為了釐清電刺激促進骨分化造成的細胞數下調是否與細胞週期停滯(Cell cycle arrest)有關聯性，分別進行細胞生長曲線(Cell growth curve)及二階細胞週期分析(Two-step cell cycle analysis)，發現電刺激組別細胞生長有趨緩，且細胞週期分析顯示CM情況下電刺激組別會造成G2M期細胞比例上升，但OM情況下則是G1期細胞比例上升。以西方墨點法分析細胞週期與訊息傳遞之蛋白質發現，CM情況下p21、p53有上調控，確認細胞週期確實有停滯，Rb之磷酸化比例與Cyclin E1上升，代表細胞加速脫離G1期。以G2/M檢查點Wee1與CDK1磷酸化之提升，和Cyclin A2之上調與Cyclin B1之下調，證實細胞週期被停滯在G2期。在OM情況下，p21亦有明顯提升，且Rb之磷酸化比例與G1期相關Cyclin D1、Cyclin E1、CDK4、CDK6下調，證實細胞在骨分化藥物和電刺激下會造成G1期停滯。而在Smad家族蛋白之結果，發現不論是CM或OM，其Smad3、Smad1/5之磷酸化比例和Smad4之表現量皆有明顯提升，推測直接電刺激藉由加快Smad訊息傳遞造成骨分化能力上調與p21表現增加使細胞週期停滯。
摘要(英) In this study, human dental pulp stem cells, hDPSC, cultured in regular culture medium (CM) or osteogenesis medium (OM) were treated direct current stimulation to discuss the effect of electrical stimulation on cell cycle and osteogenesis. The calcium deposition and cell number assay demonstrated that the mineralization of electrical stimulation treated group was improved 1.5 to 2 folds compared to the control group, however, it also reduced cell amounts. To investigate whether the reduction of cell under electrical stimulation resulted from cell cycle arrest, cell growth curve and two-step cell cycle analysis were performed. The results suggested that cell growth was retarded by electrical stimulation, and cells were arrested at G2/M and G1 when they were cultured in CM and OM, respectively. Therefore, we applied Western blotting to analyze proteins relative to cell cycle and signal transduction. In CM condition, p21 and p53 were up-regulated, suggesting cell cycle was arrested by electrical stimulation. Phosphorylation levels of Rb and Cyclin E were both increased, indicating cells were accelerated to leave from G1. The up-regulation of Cyclin A2 and down-regulation of Cyclin B1 suggested that cells were arrested at G2 stage. Therefore, we further analyzed Wee1 and phosphorylation of CDK1, the regulated protein in the checkpoint between G2 and M stages. The results demonstrated that Wee1 was up-regulated to increase the phosphorylation of CDK1, which eventually caused G2 stage arrest. In the OM condition, p21 was also unregulated. In addition, phosphorylation of Rb as well as the down regulations of Cyclin D1, Cyclin E1, CDK4, and CDK6 all supported that cell treated electrical stimulation in OM were arrested in G1 stage. Finally, the gene regulation of Smad proteins results demonstrated that Smad3 and Smad1/5 were both highly phosphorylated and the Smad4 was also up-regulated. These results indicated that electrical stimulation can accelerate Smad-mediated signal transduction, which not only improved osteogenesis but also up-regulated p21 to arrest cell cycle.
關鍵字(中) ★ 聚吡咯
★ 電刺激
★ 牙髓幹細胞
★ 骨分化
★ 細胞週期		 關鍵字(英)
論文目次 摘要 I
Abstract III
致謝 IV
目錄 V
表目錄 IX
圖目錄 X
第一章 緒論 1
1.1 研究動機 1
1.2 實驗目的 5
第二章 文獻回顧與理論基礎 6
2.1 組織工程 6
2.2 幹細胞 8
2.2.1 牙髓間質幹細胞 10
2.3 骨分化過程 12
2.3.1 SMAD 蛋白家族 (SMAD protein family) 14
2.4 細胞週期 16
2.4.1 細胞週期素 (Cyclins) 19
2.4.2 細胞週期素依賴性激酶 (Cyclin-dependent kinases, CDKs) 20
2.4.3 p53腫瘤抑制蛋白 (p53 tumor suppressor protein) 23
2.4.4 p21細胞週期素依賴性激酶抑制蛋白 (p21 cyclin-dependent kinase inhibitor protein) 24
2.4.5 Wee1 G2檢查點激酶(Wee1 G2 checkpoint kinase) 26
2.4.6 Rb視網膜母細胞瘤蛋白 (Rb retinoblastoma protein) 27
2.5 物理刺激與細胞週期 28
2.6 細胞週期延滯對細胞生理影響 33
2.6.1 細胞複製之衰老(Replicative senescence) 33
2.6.2 DNA修復 (DNA repair) 34
2.6.3 細胞凋亡 (apoptosis) 35
2.6.4 分化 (Differentiation) 37
2.7 電刺激 37
第三章 實驗材料與方法 41
3.1 實驗藥品 41
3.1.1 導電材料製備藥品 41
3.1.2 細胞培養、骨分化用藥 41
3.1.3 生長曲線定量試劑 42
3.1.4 骨分化定性、定量試劑 43
3.1.5 多光源微量自動細胞分析模組定量試劑 44
3.1.6 蛋白質萃取、定量、定性試劑 44
3.2 實驗儀器 46
3.3 試藥製備與實驗方法 48
3.3.1 Polypyrrole film與電刺激裝置製備 48
3.3.2 細胞繼代培養與冷凍、解凍 49
3.3.3 骨分化培養液配方 (Osteogenesis medium) 51
3.3.4 茜素紅染色定性分析 (Alizarin Red Staining) 52
3.3.5 Calcium-O-Cresophtalein Complexone定量分析 54
3.3.6 MTT 分析 57
3.3.7 Two-step cell cycle 分析 59
3.3.8 蛋白質收樣與定量分析 62
3.3.9 蛋白質膠體電泳(SDS-PAGE) 63
3.3.10 西方墨點分析法 (Western Blot analysis) 67
3.4 實驗架構設計 73
3.4.1 直接電刺激對細胞骨分化的影響 74
3.4.2 直接電刺激對細胞生長速率與細胞週期分布的影響 74
3.4.3 直接電刺激對蛋白質分子機制的影響 76
第四章 結果與討論 77
4.1 直接電刺激對細胞骨分化的影響 77
4.1.1 茜素紅定性分析與鈣離子沉積定量分析骨分化效果 77
4.2 直接電刺激對細胞生長速率與細胞週期分布影響 80
4.2.1 細胞生長速率 80
4.2.2 細胞週期分布 83
4.3 直接電刺激對蛋白質分子機制的影響 88
4.3.1 直接電刺激對p53及p21之影響 88
4.3.2 直接電刺激對Rb磷酸化程度的影響 92
4.3.3 直接電刺激對於G2/M期Cyclins、CDKs與Wee1的影響 95
4.3.4 直接電刺激對於G1期Cyclins與CDKs的影響 98
4.3.5 直接電刺激對於Smad家族蛋白的影響 106
第五章 結論 115
參考資料 119
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指導教授 胡威文 審核日期 2016-8-29
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