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研究生:李光遠
研究生(外文):Kuang-Yuan Li
論文名稱:小鼠胚幹細胞株於膠原蛋白基質之分化研究
論文名稱(外文):The differentiation of mouse embryonic stem cell line in collagen matrices
指導教授:黃玲惠
指導教授(外文):Lynn L.H. Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:50
中文關鍵詞:胚幹細胞膠原蛋白分化
外文關鍵詞:differentiationembryonic stem cellcollagen
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本研究是以小鼠胚幹細胞作為實驗的細胞種類,分別在二維培養及三維培養系統中,探討不同培養基成分誘導幹細胞分化的潛力,並且評估微環境對幹細胞分化所產生的影響。
在二維培養的情況下,利用多種不同的細胞培養基作為D3 cell line的養分來源,包含D3 cell culture medium (DMEM with 15% FBS) only,D3 cell culture medium with 0.5□M cyclic AMP and 10mM retionic acid,D3 cell culture medium with 20mM retionic acid,D3 cell culture medium with 20mM Ca++,M199 culture medium with 10 ng/ml bFGF,Keratinocyte growth medium等,評估不同成分對於幹細胞分化為內皮細胞、表皮細胞與神經細胞的影響。除此之外,我們改變細胞培養的條件,如在培養盤表面塗布一層膠原蛋白,以探討微環境對幹細胞生長與分化造成的效應。研究結果顯示在D3 cell culture medium only培養情況下,我們可得到較多數目的內皮細胞。
在三維培養的情況中,以磁珠分離的方式獲得在二維培養下已分化之內皮細胞,並且利用本實驗室所研發之豬源多孔性膠原蛋白基質做為培養材料,進一步將人類纖維母細胞與內皮細胞共同培養在多孔性膠原蛋白基質中。透過免疫組織化學染色的方式,我們觀察到有類微血管結構的生成,以及發現內皮細胞會表達出其分子標誌CD34與vWF。其所形成的管狀結構周圍,亦有明顯的膠原蛋白第四型分子的生成。
Using the mouse embryonic stem cells (ES cells), we demonstrated the effects of culture medium with different supplements on induction potentials in 2-D and 3-D cultural systems and also evaluated the influences of microenvironments on cell differentiation.
In two dimensional cultivation, we used several culture mediums to be the nutrient supply including D3 cell culture medium (DMEM with 15%FBS) only, D3 cell culture medium with 0.5□M cyclic AMP and 10mM Retionic acid, D3 cell culture medium with 20mM retionic acid, D3 cell culture medium with 20mM Ca++, M199 culture medium with 10ng/ml bFGF, and Keratinocyte growth medium etc., to evaluate the influences on the differentiation of stem cells into endothelial cells, keratinocytes and neurons. Besides that, in order to study the effects of microenvironments on the growth and differentiation of ES cells, we changed the microenvironments on cell cultural conditions, such as coating collagen on the culture plate surface. The Results indicated that we obtained more endothelial cells by using D3 culture medium only.
In three dimensional cultivations, we separated endothelial cells by the way of magnetic beads from differentiated cell populations and used porcine porous collagen matrices as cultural materials, then co-cultured human fibroblasts with endothelial cells in the matrices. Through the immunohistochemical staining, we observed that the micro capillary-like tube formation and found that endothelial cells expressed CD34 and vWF, the molecular markers of endothelial cells. In the tube surroundings, there were also significant signals of type IV collagen depositions.
目 錄
中文摘要
英文摘要
目錄
表目錄
圖目錄
第一章 緒論1
第二章 文獻回顧6
2.1 幹細胞的特性6
2.2幹細胞的分化機制及其分化方向的限制8
2.3自動物體內分離並建立幹細胞株11
第三章 實驗儀器、藥品、方法14
3.1 實驗藥品14
3.2 實驗儀器16
3.3 實驗方法18
3.3.1 D3-ES cells的細胞培養18
3.3.2飼養層的製備19
3.3.3 D3-ES cell多功能性測試19
3.3.4 Embryoid Body formation19
3.3.5以不同成分培養基在二維環境下培養D3-ES cells20
3.3.6 以不同成分的培養基在三維培養環境下培養D3-ES cells20
3.3.7在不同微環境中培養D3-ES cells20
3.3.8 自幹細胞分化群中分離出內皮細胞22
3.3.9 三維共培養系統22
3.3.10塑膠切片製備23
3.3.11冷凍切片製備23
3.3.12病理染色(H&E staining)24
3.3.13免疫組織化學染色(immunohistochemical staining)24
3.3.14以流式細胞儀進行細胞分化的定量24
第四章 結果討論26
第五章 實驗結論29
第六章 參考文獻30

表 目 錄
表1.1 目前已知幹細胞之來源38
表1.2 老鼠胚胎幹細胞在in vitro的環境下可以分化的細胞類型40
表2.1 常用於辨識幹細胞及判斷細胞分化的標幟分子 41
表3.3.1不同成分的培養基在二維培養環境下對於D3-ES cell分
化行為之影響45
表3.3.2不同微環境(microenvironment)對於D3-ES cell分化
成內皮細胞之影響47
表3.3.3不同成分的培養基在三維培養環境下對於D3-ES cell分
化行為之影響48
表3.3.4 自已分化之細胞群中分離出內皮細胞細細胞進行三維
立體培養49

圖 目 錄
圖1.1 目前幹細胞研究之應用36
圖2.1 受精卵發育的過程36
圖2.2 人體胚胎幹細胞萃取的流程37
圖2.3 標幟分子結合FACS技術以分別細胞37
圖2.4 螢光標的方法39
圖3.3.1 alkaline phosphatase assay測試結果42
圖3.3.2 在細胞培養盤中存在有許多不同型態的細胞42
圖3.3.3 在懸滴培養系統中得到(A)簡易胚胎體與(B)囊狀胚胎體43
圖3.3.4 血管成熟度之分子鑑定(A)CD34 (B)vWF (C)type IV collagen50
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