凝膠濃度對胎盤幹細胞貼附及分化之影響

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姓名

曹昌雅(Chung-ya Tsao) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

凝膠濃度對胎盤幹細胞貼附及分化之影響
(Gelatin Concentration Affect the Attachment and Differentiation of Placenta-Derived Multi-Potent Cells)

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摘要(中)

本研究主要以製備生物相容性之含水凝膠(Gelatin)做為培養人類胎盤幹細胞(Placenta-derived multi-potent cells, PDMCs)之基材。而粉末狀凝膠分為酸性及鹼性，可經由調整水和粉末的比例來控制不同重量百分濃度，本實驗以6%、8%、10%、12%、14%和16%之凝膠水溶液和膠聯劑－戊二醛，依固定比例均勻混和，來製備不同重量百分濃度之凝膠基材。凝膠在製備完成後，以熱重分析儀來檢測其重量百分濃度與凝膠澎潤現象。透過熱微差掃描分析儀(Differential canning calorimetry)來測量粉末狀凝膠有玻璃轉化溫度與熔點，但經由戊二醛膠聯後則沒有玻璃轉化溫度與熔點。
將PDMCs種植在凝膠基材上，並且經由細胞染色計算細胞貼附率。在酸性及鹼性凝膠基材細胞的貼附率皆在70%以上，說明此凝膠基材的表面適合PDMCs的生存；另外經由顯微鏡連續拍攝之影像，透過影像處理可以計算出PDMCs生長在凝膠基材上細胞面積的變化，其中在酸性及鹼性12%凝膠基材，細胞的面積有較明顯的變化。
另外以不同濃度凝膠為基材來誘導PDMCs分化成似神經細胞，細胞貼附在不同濃度的凝膠表面有不同的附著力(Focal adhesion)，此為環境刺激；以3-Isobutyl-1-methylxanthine (IBMX)為化學刺激，進而探討在細胞懸浮狀態下的PDMCs與IBMX共同培養在不同重量百分濃度凝膠兩小時之貼附率與分化率。將此實驗與傳統的方式比較，發現在細胞懸浮狀態下就以IBMX誘導使細胞分化為似神經細胞之方法的分化效果較好。
因為不同濃度凝膠造成不同的分化率，因此在最後將PDMCs種植在不同重量百分濃度凝膠基材上，其細胞和不同基材間也有不同的附著力(Focal adhesion)，造成細胞骨架排列及產生不同的細胞膜蛋白質，在實驗的最後利用一維電泳的方式來證明細胞貼附在不同重量百分濃度之凝膠基材上，產生出不一樣的細胞膜蛋白質。

摘要(英)

This study is mainly in preparation of biocompatible hydrogel－gelatin as a substratum to culture placenta-derived multi-potent cells (PDMCs). There are acidic and basic powder forms of gelatin. They easy to control different weight percentage by adjusting the mixture weight percentage of gelatin. There is glass transition temperature in powder form gelatin. But glass transition temperature disappeared when different weight percentage of gelatin solutions (6%, 8%, 10%, 12%, 14% and 16%) are cross-linked by glutaraldehyde, due to gel formation. The results can be measured by differential scanning calorimetry (DSC). And water content of different weight percentages of gelatin can be measured by thermogravimetry analysis (TGA).
Attachment ratio is calculated by nucleus staining when cell seeding overnight. It illustrated that the surfaces of the substrate are appropriate for PDMCs adhesion. We also calculated contact area of PDMCs adhered on hydrogel by continuous image capture by microscopy.
When PDMCs adhered on different weight percentages of gelatin, there are different focal adhesions in each other, which is an environmental stimulation. And using 3-Isobutyl-1-methylxanthine (IBMX) to induce PDMCs differentiation into neuronal-like cell is chemistry stimulation. PDMCs was treated IBMX as suspension for two hours before seeding, and the differentiation ratio was evaluated when cell has adhered on substrate.
PDMC adhered on different substrates caused different arrangement of the cytoskeleton and produced different membrane proteins. Therefore electrophoresis was used to distinguish different membrane protein production when PDMCs adhered on different substrates.

關鍵字(中)

★ 凝膠
★ 分化
★ 細胞膜蛋白質
★ 胎盤幹細胞

關鍵字(英)

★ Placenta-derived multi-potent cells
★ membrane proteins
★ differentiation
★ gelatin

論文目次

摘要 II
Abstract III
致謝 IV
目錄 V
圖目錄 VII
表目錄 XI
第一章緒論 1
1.1 前言 1
1.2 文獻回顧 1
第二章凝膠的製備 5
2.1 凝膠製備方法 5
2.2 熱重分析儀分析凝膠 8
2.3 熱微差掃描分析儀 14
第三章 Placenta-derived multi-potent cells (PDMCs)的培養 19
3.1 顯微鏡系統 19
3.2 細胞培養 21
3.3 PDMCs之貼附率分析 24
3.4 PDMCs接觸面積分析 28
第四章誘導PDMCs分化為似神經細胞 37
4.1 細胞懸浮液與IBMX混合誘導PDMCs分化為似神經細胞 38
4.2 細胞貼附12小時後以IBMX誘導PDMCs分化為似神經細胞 42
第五章 SDS-PAGE分析PDMCs表面蛋白質 46
5.1 一維電泳 47
結論 49
參考文獻 50
附件一明膠製備過程與使用之材料及設備 54
附件二細胞培養與染色之方法及使用的材料設備和配方 56
附件三偵測細胞邊界程式 63
附件四誘導PDMC分化成神經的材料及設備 65
附件五 PDMCs細胞膜蛋白質之材料和實驗步驟 66

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指導教授

李泉(Chuan Li)

審核日期

2012-2-2

推文