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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Title:	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
Authors:	陳敦祈;Chen,Tun-chi
Contributors:	化學工程與材料工程學系
Keywords:	聚?咯薄膜;電刺激;成骨;間質幹細胞;骨分化基因;polypyrrole;electrical stimulation;osteoblast;stromal stem cell;osteoblast-related gene
Date:	2013-08-19
Issue Date:	2013-10-08 15:07:20 (UTC+8)
Publisher:	國立中央大學
Abstract:	本研究以導電性高分子聚?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。 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.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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