直接電刺激對於人類牙髓幹細胞在骨分化過程中基因調控與分化能力影響之研究

The Effects of Direct Electrical Stimulation on Gene Regulation and Differentiation of Human Dental Pulp Stem Cells During Osteogenesis Process

郭弘偉(Hong-wei Guo)

指導教授 ： 胡威文

國立中央大學/工學院/化學工程與材料工程學系/碩士(2015年)

摘要

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。

關鍵字

聚吡咯 ； 電刺激 ； 成骨細胞 ； 牙髓幹細胞 ； 成骨相關基因

並列摘要

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.

並列關鍵字

polypyrrole ； electrical stimulation ； osteoblast ； hDPSC ； osteoblast-related gene

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