以電刺激增進骨髓基質細胞骨分化之最佳化探討

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陳敦祈(Tun-chi 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

以電刺激增進骨髓基質細胞骨分化之最佳化探討
(Optimization of Electrical Stimulation for Improving Osteogenesis of Bone Marrow Stromal Cells)

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摘要(中)

本研究以導電性高分子聚吡咯薄膜做為培養大鼠骨髓間葉幹細
胞的基材，並經由骨分化時期不同時間電刺激與調控電刺激物理參數
以觀察幹細胞成骨分化的影響。首先在不同時間電刺激方面，分別在
更換骨分化培養液後作為第0, 2, 4, 6, 8, 10, 12 天個別對細胞進行一
次4 小時的恆定電壓刺激。再於第14 天進行茜素紅染色及鈣沉積定
量分析。結果發現，於骨分化第8 天電刺激的組別與其他時間點電刺
激相比有較明顯的礦化現象。利用RT PCR 及qPCR 結果來分析骨相
關基因調控，發現電刺激可以延長ALP 及Cbfa1 的表現時間，增加
osteocalcin 基因的表現，並加速osteopontin 基因的下降。接著以骨分
化培養第8 天電刺激為最佳時間點，分別調控電刺激參數，並於第
14 天進行茜素紅染色以及鈣沉積定量分析。首先與定電壓相比，0.01
Hz 方波具有較佳骨化效果。另外在調整振幅及偏移中，發現決定骨
化的效果主要是在於實質上的通電電壓而非振幅。最後我們以不同的
運作時間比來調控方波，發現運作時間比介於10%至99%效果最佳。

摘要(英)

Polypyrrole, was applied to culture rat bone marrow stromal cells
(BMSCs) to determine the effect of the period of electrical stimulation
(ES) and parameters of electricity on osteogenesis. For period of ES
experiments, BMSCs were induced using osteogenic and were treated
constant 2V at Day 0, 2, 4, 6, 8, 10, 12. Alizarin red staining and calcium
deposition analysis at Day 14 were performed, which demonstrated that
ES at Day 8 led to highest mineralization. Therefore,
reversed-transcription PCR and quantities PCR were applied to analyze
the osteogenic relative gene regulation. The results indicated that ES at
Day 8 can not only extend the period of alkaline phosphatase and Cbfa1
genes, but also accelerate the promotion of osteocalcin gene and the
decline of osteopontin gene. Then we regulated different ES parameters
and evaluated using alizarin red staining and calcium deposition analysis
at Day 14. Compared to constant voltage, square waves with 0.01 Hz may
improve the level of calcium deposition. Through adjusting the amplitude
and offset, the effect of operation voltage should be more important than
the amplitude. Finally, different duty cycles were applied to regulate
square waves, and the results suggested that 10% to 99% of duty cycles
demonstrated optimal mineralization.

關鍵字(中)

★ 聚吡咯薄膜
★ 電刺激
★ 成骨
★ 間質幹細胞
★ 骨分化基因

關鍵字(英)

★ polypyrrole
★ electrical stimulation
★ osteoblast
★ stromal stem cell
★ osteoblast-related gene

論文目次

摘要................................................................................................................................ I
Abstract ......................................................................................................................... II
致謝.............................................................................................................................. III
目錄.............................................................................................................................. IV
圖表目錄...................................................................................................................... VI
第一章緒論........................................................................................................ 1
1-1 研究動機................................................................................................ 1
1-2 實驗假說................................................................................................ 3
第二章文獻回顧與理論基礎............................................................................ 4
2-1 組織工程................................................................................................ 4
2-2 幹細胞.................................................................................................... 6
2-3 骨分化進程與骨相關基因.................................................................... 8
2-4 電刺激.................................................................................................. 10
第三章實驗方法與設備.................................................................................. 16
3-1 實驗藥品.............................................................................................. 16
3-1-1 材料製備用藥.............................................................................. 16
3-1-2 細胞培養、骨分化用藥.............................................................. 16
3-1-3 骨分化定性、定量試劑.............................................................. 19
3-1-4 Total RNA 萃取、反轉錄cDNA、PCR 試劑、膠體電泳 ....... 20
3-2 實驗儀器.............................................................................................. 23
3-3 試藥製備與實驗方法.......................................................................... 25
3-3-1 Polypyrrole 製備、電刺激裝置製作 .......................................... 25
3-3-2 細胞冷凍解凍、培養繼代.......................................................... 27
3-3-3 不同時間點電刺激實驗、電刺激參數最佳化.......................... 28
3-3-4 茜素紅染色(Alizarin Red Staining, ARS) .................................. 32
3-3-5 Calcium-O-Cresolphtalein Complexone ...................................... 33
3-3-6 Purification of Total RNA from Animal Cells Using Spin
Technology-RNeasy Mini Kit ...................................................................... 35
3-3-7 SuperScript III First-Strand Synthesis System for RT-PCR ........ 36
3-3-8 Polymerase chain reaction (PCR) & Electrophoresis .................. 38
3-3-9 Real-Time PCR (Quantitative Polymerase Chain Reaction) ....... 41
3-4 實驗設計與架構.................................................................................. 44
3-4-1 導電高分子聚吡咯親疏水性與電性質...................................... 44
V
3-4-2 不同時間點電刺激對骨分化的影響.......................................... 44
3-4-3 最佳化電刺激參數增進骨分化現象.......................................... 45
3-5 材料物理性質分析.............................................................................. 47
3-5-1 親疏水性分析.............................................................................. 47
3-5-2 導電性分析.................................................................................. 48
3-5-3 穩定性測試.................................................................................. 49
3-6 生物性質分析...................................................................................... 50
3-6-1 茜素紅染色(Alizarin Red Staining, ARS) .................................. 50
3-6-2 Calcium-O-Cresolphthalein Complexone .................................... 50
3-6-3 骨分化相關基因表現.................................................................. 51
第四章結果與討論.......................................................................................... 61
4-1 導電高分子聚吡咯.............................................................................. 61
4-1-1 親疏水性(Wettability) ................................................................. 61
4-1-2 電性質量測(Conductivity) .......................................................... 62
4-1-3 穩定性測試(Stability) ................................................................. 66
4-2 不同時間點電刺激之骨分化表現...................................................... 67
4-2-1 茜素紅染色與鈣離子沉積分析.................................................. 67
4-2-2 Total RNA 萃取、反轉錄PCR 和膠體電泳 ............................. 74
4-2-3 即時聚合脢連鎖反應(Real-Time PCR) ..................................... 77
4-3 最佳化電刺激參數.............................................................................. 82
4-3-1 茜素紅染色與鈣離子沉積分析.................................................. 82
第五章結論...................................................................................................... 89
第六章參考資料.............................................................................................. 91

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指導教授

胡威文(Wei-wen Hu)

審核日期

2013-8-19

推文