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研究生:江俊儀
研究生(外文):ChunYi Chiang
論文名稱:應用直流電場促進體外培養之骨髓基質細胞增殖
論文名稱(外文):Application of Direct Current Electric Fields Promote Proliferation of Bone Marrow Stromal Cells in vitro
指導教授:黃勇三
指導教授(外文):YongSan Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:70
中文關鍵詞:骨髓基質細胞直流電場細胞增殖細胞內訊息傳遞
外文關鍵詞:bone marrow stromal celldirect current electric fieldcellular proliferationintracellular signal transduction
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電場及電磁場可促進成骨及骨母細胞之增殖,因此目前已被運用於治療骨科疾病,也有報告指出電場及電磁場可促進受損的周邊及中樞神經系統之軸突再生。然而電場刺激對幹細胞的影響仍不清楚,因此本實驗使用自骨髓中分離出之骨髓基質細胞做為多潛能幹細胞之模式。已有許多報告指出骨髓基質細胞擁有跨胚層分化的能力,除了可分化成多種不同的中胚層細胞外,還可分化為神經元、肺泡上皮、胸腺細胞等;故本實驗應用不同強度之直流電場刺激骨髓基質細胞,並在不同的時間點觀察直流電場對細胞之增殖能力、存活能力及細胞內訊息傳遞之影響。細胞經電場連續刺激48小時後,利用BrdU標定,配合免疫化學染色、流式細胞儀及非放射性細胞生長偵測套組(MTS)偵測骨髓基質細胞數目及細胞週期分佈,結果發現24V/3cm或35V/3cm強度的電場刺激48小時後,與對照組相比細胞數顯著性增加(p < 0.05),表示這兩種強度的電場皆可促使骨髓基質細胞增殖;為瞭解電場對細胞內訊息傳遞之影響為何,故挑選與細胞生長增殖相關的訊息傳遞路徑予以抑制。結果發現給予Tyrosine磷酸化抑制劑或MEK kinase抑制劑皆可阻斷電場刺激細胞增殖的能力。因此再利用SDS膠體電泳及西方墨點法偵測MEK下游之ERK訊息蛋白,結果發現電場刺激在5到7分鐘內即可明顯提升細胞內ERK磷酸化蛋白之表現量,可知電場可能會藉由活化MEK/ERK的訊息傳遞路徑而促使骨髓基質細胞的增殖。而由以上實驗結果,我們期望日後能將電刺激合併移植骨髓基質細胞至中樞神經受損之動物體,以其加強骨髓基質細胞移植後之存活及促進軸突再生的能力。
Electric and electromagnetic fields have already been applied for therapy of bone disease at present because of promoting proliferation of osteocytes and osteoblasts. Some studies also suggested that these treatments can promote axon regeneration in injuried peripheral and central nervous system. However, how these applications effects on stem cells are unknown. Therefore, we choose bone marrow stromal cells (BMSCs) which were separated from bone marrow cells as a model of the multipotential stem cell. There are many reports claimed that BMSCs could not only differentiate into other mesenchymal cells but also trans-differentiate into non-mesenchymal cells, such as neurons, alveolar epithelial cells and thymus cells. In the present studies, we have investigated the effects of direct current (DC) electric fields on cellular proliferation, cellular viability, and signal transduction of BMSCs for different periods in vitro. By using immunochemical staining method, MTS assay and flow cytometry, we found that cell numbers of BMSC exposed to 24V/3cm or 35V/3cm DC electric fields for 48 h were significantly higher than those of controls. In order to investigate intracellular signal transduction, we used inhibitors which correlate with cell proliferation. This result indicates that treatment with either Genistein (protein tyrosine kinase inhibitor) or U0126 (MEK inhibitor) blocks the effect of DC electric fields. Then we detected intracellular phosphor-ERK 1/2 by SDS-PAGE and Weston blot. The result of western blot shows that phospho-ERK 1/2 raised from 5-7 min after DC electric fields treated. It means that DC electric fields seem to promote proliferation of BMSCs dependence on MEK/ERK pathway. We hope the application of DC electric fields combined with BMSCs transplantation in vivo to enhance cells viability and proliferation in the future.
中文摘要.............................................................Ⅰ
英文摘要.............................................................Ⅱ
縮寫表...............................................................Ⅲ
圖次................................................................Ⅴ

第一章 緒言.........................................................1
第二章 文獻探討......................................................2
第一節 電、電場與電磁場...............................................2
第二節 電場及電磁場對生物之影響........................................5
2-2.1 治療骨科疾病....................................................5
2-2.2 促進骨骼修復與重塑之機制..........................................7
影響鈣離子通道........................................................8
影響跨細胞膜受器.......................................................9
影響傳訊RNA...........................................................9
合成生長因子.........................................................10
2-2.3 對其他細胞影響.................................................11
免疫方面.............................................................11
神經方面.............................................................12
2-2.4 致癌性與致畸胎之流行病學調查.....................................12
第三節 細胞增殖之調控...............................................14
2-3.1 與細胞增殖相關的訊息傳遞路徑.....................................15
Mitogen-activated protein kinase (MAP kinases)....................15
Phosphatidylinositol 3-kinase (PI3K)..............................16
第四節 骨髓基質細胞..............................................18
第三章 材料與方法...................................................21
第一節 骨髓基質細胞.................................................21
3-1.1. 骨髓基質細胞之培養.............................................21
3-1.2. 骨髓基質細胞之純化.............................................22
3-1.3. 骨髓基質細胞之純度評估方法與分析.................................22
第二節 直流電場刺激骨髓基質細胞存活及增殖能力之測定.....................23
3-2.1. 直流電場裝置..................................................23
3-2.2. 骨髓基質細胞各時間點分裂能力與電壓關係觀察........................23
3-2.3. 計數與統計分析方法.............................................25
3-2.4. 細胞生長活性測試...............................................25
3-2.5. 免疫螢光染色之步驟.............................................26
第三節 生長週期細胞相分佈測定........................................26
3-3.1. 樣品收集......................................................26
3-3.2. 流式細胞儀分析................................................27
第四節 訊息傳遞抑制劑拮抗電場作用試驗.................................28
第五節 電場影響細胞內訊息傳遞測定.....................................28
3-5.1. 樣品收集......................................................28
3-5.2. 蛋白質濃度測定................................................29
3-5.3. SDS-聚丙烯醯胺膠體電泳法.......................................29
配製10%聚丙烯醯胺的分離膠體溶液.......................................29
配製3.6%聚丙烯醯胺的Stacking gel.....................................30
蛋白質電泳...........................................................30
3-3.4. 西方墨點法....................................................30
第四章 實驗結果.....................................................32
第一節 骨髓基質細胞培養及純化結果.....................................32
4-1.1 骨髓基質細胞培養................................................32
4-1.2 骨髓基質細胞之純化結果..........................................32
第二節 直流電場刺激對骨髓基質細胞增殖之影響............................33
4-2.1 骨髓基質細胞各時間點分裂能力與電壓關係............................33
4-2.2 電場刺激細胞生長活性............................................33
4-2.3 BrdU標定細胞之標準量化.........................................34
4-2.4 生長週期細胞相分佈..............................................34
第三節 直流電場刺激對細胞內訊息傳遞之影響..............................35
4-3.1 訊息傳遞抑制劑拮抗電場作用.......................................35
Genistein (protein tyrosine kinase inhibitor).....................35
LY294002 (PI3 kinase inhibitor)...................................36
U0126 (MEK kinase inhibitor)......................................36
4-3.2 西方墨點法.....................................................37
第五章 討論........................................................49
參考文獻.............................................................55
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