在細胞增殖相關的訊息傳導中，Notch1訊息傳遞路徑扮演重要的腳色。其信號產生是通過相鄰細胞的配體和受體結合，裂解出Notch1胞內結構域(N1IC)進入細胞核激活下游基因調節。為了研究增強Notch1胞內結構域是否可影響細胞增生，本研究將不同長度之Notch1胞內結構域與GST融合的蛋白質結合於GSH表面修飾的磁性奈米顆粒上，利用瞬間磁場的作用轉導進入細胞，以增強Notch1路徑之訊號。由實驗室前人之實驗結果得知經轉導N1IC融合蛋白質之間質幹細胞，培養兩天後與對照組相比增生率有明顯的上升。因此進 一步針對增生路徑上之相關基因與蛋白質的表現進行RT-PCR和西方墨點法檢測。結果顯示轉導N1IC之間質幹細胞的HES1基因表達增強，PTEN基因表達量下降，且磷酸化Akt蛋白質的表現量增加，並且仍可以分化成脂肪細胞。由此結果可確認轉導N1IC可加強間質幹細胞之增生途徑上PI3K-AKT路徑的活化，促使間質幹細胞增生，但增生後之細胞仍保有分化能力。

In cell proliferation signals, Notch1 signal pathway plays a very important role. The Notch 1 signal is generated via the binding of ligands and receptors of adjacent cells, and then cleaves out the Notch1 intracellular domain (N1IC) into the nucleus to activate downstream gene regulation.
In order to investigate whether enhancing the Notch1 intracellular domain can induce cell proliferation, this research carried different lengths of the Notch1 intracellular domain and proteins fusion with GST mark on the surface of the GSH-modified magnetic nanoparticles and transfected into cells by generating an instantaneous magnetic field in order to increase the signal to activate the Notch1 pathway. According to the experiment result from senior student knows that transfect N1IC fusion protein into mesenchymal stem cells, after two days culturing, the proliferation rate increased significantly compared with the control group. Therefore, the performance of related genes and proteins on the Notch1 sign pathway was further tested by RT-PCR and Western blot. The result shows that transfect N1IC can upregulation the expression of HES1 gene, downregulation the PTEN gene and increase the expression of phospho-Akt, and still can differentiate into Adipogenesis cells. From these results, it can be confirmed that transfection of N1IC can influence the genes HES1, PTEN and PI3K-AKT pathway in the proliferation pathway of mesenchymal stem cells, and the cells still retain the differentiation ability after transfection.

摘要 1
Abstract 2
目錄 4
圖目錄 9
表目錄 11
第一章、緒論 12
1.1、前言 12
1.2、研究動機 13
第二章、文獻回顧 14
2.1、超順磁氧化奈米顆粒 14
2.2、間質幹細胞 16
2.3、Notch訊號 17
2.3.1、Notch受體 18
2.3.2、Notch配體 19
2.4、Notch增生路徑 20
2.4.1、轉錄因子CSL 21
2.4.2、Hes1 22
2.4.3、磷酸酯酶與張力蛋白同源物 23
2.4.4、PI3K-Akt路徑 24
2.5、奈米磁性顆粒製備與應用於轉導蛋白質進入細胞 26
第三章、實驗藥品及設備 27
3.1實驗藥品 27
3.1.1、實驗細胞來源 27
3.1.2、實驗菌種來源 28
3.1.3、菌種培養基 28
3.1.4、蛋白質分析 28
3.1.5、蛋白質定量 29
3.1.6、RT-PCR 29
3.1.7、奈米磁顆粒製備 30
3.1.8、西方墨點法(Western Blot) 31
3.1.9、細胞培養與磁衝 31
3.1.10、成脂分化 32
3.2、實驗設備 32
第四章、實驗步驟 35
4.1、實驗流程 35
4.2、SPION@SiO2-MA-GSH製備 36
4.2.1、超順磁氧化奈米顆粒(SPION) 36
4.2.2、SPION@SiO2 37
4.2.3、SPION@SiO2-NH2 37
4.2.4、SPION@SiO2-MA 38
4.2.5、SPION@SiO2-MA-GSH 38
4.2.6、Ninhydrin reaction 39
4.3、不同長度之Notch1胞內結構域蛋白質製備 40
4.3.1、產不同長度Notch1胞內結構域之重組菌培養 40
4.3.2、取得蛋白質粗萃液 41
4.3.3、重組菌保存 41
4.4、GST融合蛋白質固定 41
4.5、磁衝轉導間質幹細胞 43
4.5.1、間質幹細胞培養 43
4.5.2、間質幹細胞分盤 43
4.5.3、間質幹細胞保存 44
4.6、轉導間質幹細胞 44
4.7、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS PAGE) 46
4.7.1、待測樣品前處理與膠體配置 46
4.7.2、電流條件 48
4.8、逆轉錄聚合酶鏈式反應(reverse transcription-PCR) 49
4.8.1、自細胞分離RNA 49
4.8.2、RNA反轉錄為cDNA 50
4.8.3、聚合酶連鎖反應(Polymerase chain reaction) 50
4.9、西方墨點法(Western Blot) 51
4.9.1、細胞樣品前處理 52
4.9.2、西方墨點法步驟與電流條件 52
4.10、脂肪細胞分化 55
第五章、結果與討論 56
5.1、重組大腸桿菌表達融合GST之Notch1胞內結構域 56
5.2、固定GST-融合蛋白質 61
5.3、逆轉錄聚合酶鏈式反應(reverse transcription-PCR) 63
5.4、西方墨點法結果分析 69
5.5、分化能力測試 71
5.6、綜合討論 74
第六章、結論與建議 78
6.1、結論 78
6.2、建議 78
第七章、參考文獻 80

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官予馨“藉由轉導Notch1細胞內結構鍵結磁性奈米顆粒進入人類間
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陳彥榮“多能性幹細胞 ”科學月刊5月號 (2014)

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