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研究生:吳雅婷
研究生(外文):Ya-Ting Wu
論文名稱:小鼠胚幹細胞衍生之雙軸突運動神經元
論文名稱(外文):Bi-axonal Aberration in Mouse Embryonic Stem Cell-DerivedMotoneurons
指導教授:蘇鴻麟蘇鴻麟引用關係
指導教授(外文):Hong-Lin Su
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:胚幹細胞運動神經元軸突
外文關鍵詞:embryonic stem cellmotoneuronaxon
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中樞神經系統中,運動神經元以乙醯膽鹼做為神經傳遞物質,負責傳
遞運動訊息到目標肌肉,在形態上呈現一條十分細長的軸突及許多分支狀的樹突。在胚胎神經發育時期,sonic hedgehog (SHH) 透過活化basic helix-loop-helix (bHLH) 蛋白質Olig2,同源結構區蛋白質Nkx6.1、Lhx3及HB9 表現,觸發運動神經元的形成。因此本研究以綠色螢光蛋白基因置HB9 啟動子下游所構成之轉殖基因,建立的小鼠胚幹細胞做為研究對象,利用stromal cell-derived inducing activity (SDIA) 神經誘導方式並以50 ng/ml SHH 與0.1μM retinoid acid (RA) 之處理,誘導小鼠胚幹細胞分化,並藉由綠色螢光蛋白質表現,以追蹤運動神經元形成。經過SDIA 誘導後,表現綠色螢光蛋白質之神經元同時具有運動神經元相關標誌choline acetyltransferase (ChAT) 、vesicular acetylcholine transporter (VAchT) 及運動神經元專一性指標蛋白質MNR2 的表現。值得注意的是,經誘導分化後,部分運動神經元相較於單一細長軸突之正常神經形態,顯示出雙重軸突之不正常形態。進一步探究後, 發現與軸突形成相關之Phosphoinositide-3-kinase (PI3K) 訊號路徑下游蛋白質Akt 及mitogen-activated protein kinas(MAPK) 訊號路徑下游蛋白質ERK,於細胞內接近軸突之位置處有累積情況,但是牽涉軸突特化的蛋白質Par3 在分化後運動神經元之軸突末梢並沒有被偵測到,因而推測分化後運動神經元的軸突形成路徑與海馬迴神經元軸突發育所透過的Partitioning defective 3/Partitioning defective 6/atypical protein kinase (Par3/Par6/aPkc) 路徑並不一致。使胚幹細胞體外分化衍生之運動神經元呈現正常發育型態,對於臨床應用於運動神經元病變或脊椎傷害之治療可助於正確神經訊息傳遞並減少錯誤的神經連結,進而改善因運動神經元損傷所導致之病症。由於本研究分化產生之雙重軸突的運動神經元可能導致樹突接收調節訊號的功能喪失,造成運動訊息的錯誤傳遞,因此目前仍不適用於移植治療應用。
Motoneurons coordinate motor functions by release of acetylcholine neurotransmitter to targeting muscles. In vertebrate central nerve system, motoneuron is featured with one extreme long, thin axon and many branched dendrites. During embryo neurogenesis, SHH patterns the ventral neural tube and induces the expression of a bHLH protein, Olig2, and homeodomain proteins including Nkx6, Lhx3 and HB9, to trigger the motoneuron formation. Here, we combined the neural inducing method “stromal cell-derived inducing activity (SDIA)” with 50 ng/ml SHH and 0.1μM RA treatment from day 4 to day 9 to enforce the differentiating mouse embryonic stem (ES) cells into motoneurons in spinal cord. HB9-GFP-transgenic ES cells, in which expression of enhanced green fluorescent protein(eGFP) is controlled under mouse HB9 promotor, was subjected to trace the ES cells-differentiating motoneurons. Induced GFP-positive neurons showed the expression of ChAT, VAchT and motoneuron specific marker, MNR2. Notably, distinguishing differentiating motoneurons from ES cells exhibited “bi-polar” axons, conflict to the normal uni-axon neural morphology of motoneurons. Especially, PI3K signalling downstream protein Akt and MAPK cascade downstream kinase, ERK, were both accumulated at the site near the axon in cell body of differentiating motoneurons, but the axon polarizing protein, Par3, was not
detected in tip of axon, suggesting that the axon formation of differentiated motoneurons may not be similar to the Par3/Par6/aPKC pathway in hippocampal neurons. Shaping the ES cells-derived motoneurons in a correct morphology may render a more correct targeting and reduces extraordinary
neural linkage for the engrafted neurons in patients with motoneuron degeneration diseases or spinal cord injury. The formation of bi-axonal motoneurons in this study may loss regulatory input signals through dendrites and transmit aberrant output stimuli to target cells while neural signals
transmission. Therefore, it is still improper cell-therapy source in clinical transplantation now .
中文摘要..........................................i
英文摘要 ........................................ ii
目 錄 ........................................... iii
圖表目錄 .........................................iv
附錄目錄 .........................................vi
第一章、 前言......................................1
第二章、 材料與方法 ...............................9
第三章、 結果......................................15
第四章、 討論......................................22
第五章、 參考文獻 .................................27
圖表結果 ......................................... 34
附錄圖 ............................................70
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