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研究生:黃琬婷
研究生(外文):Wan-Ting Huang
論文名稱:以 STZ-NA 誘發糖尿病小鼠模型探討人類脂肪間質幹細胞於第二型糖尿病治療之應用
論文名稱(外文):Investigating the fate of human adipose-derived stem cells on the treatment of type II diabetes by STZ-NA-induced diabetic mice
指導教授:邱紫文邱紫文引用關係
指導教授(外文):Tzyy-Wen Chiou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
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:2014
畢業學年度:102
論文頁數:51
中文關鍵詞:人類脂肪間質幹細胞第二型糖尿病細胞追蹤
外文關鍵詞:human adipose-derived stem cellstype II diabetescell tracing
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因生活習慣及飲食習慣改變,罹患糖尿病之患者年年趨增,其中以第二型糖尿病人數最多,占糖尿病患者總人數 95% 以上。目前能有效治療第二型糖尿病的藥物有限。源自低侵入性手術之脂肪抽取物的脂肪間質幹細胞除了具備良好的再生及分化能力,其免疫調節相關之細胞激素表現量亦頗高。本實驗探討人類脂肪間質幹細胞分化為類胰島 β 細胞之可行性,和以 STZ-NA 誘發糖尿病小鼠模式探討人類脂肪間質幹細胞移植於第二型糖尿病之效果及追蹤。研究中利用三種不同分化策略證實人類脂肪間質幹細胞具有分化為類胰島 β 細胞之潛力;而其中又以共培養與分化培養基複合式分化策略之分化效果最佳。由動物實驗得知,藉由螢光染劑(CM-Dil)標定人類脂肪間質幹細胞,可以追蹤移植細胞於化學誘導第二型糖尿病小鼠之去向。於小鼠尾靜脈注射植入人類脂肪間質幹細胞一個月後,由組織切片觀察得知植入之細胞多發現於胰臟及肝臟組織中,且細胞移植後可改善胰臟組織浸潤及胰島小球嚴重破壞情形。綜合以上結果,人類脂肪間質幹細胞除了具有分化為類胰島 β 細胞能力外,於治療第二型糖尿病亦有極大發展潛力。
Due to major changes in lifestyle and diet an increasing number of the population are becoming diabetic. Among the four types of diabetes, the type II diabetes is the most prevalent with 95% of all diabetes around the world; however, its effective treatment is still an unmet medical need.Adipose-derived stem cells can be easily obtained from donors using fat extraction by minimally invasive surgery. They are highly proliferative, multipotent and able to regulate immune and inflammatory responses.The objective of this study is to investigate the ability of human adipose-derived stem cells (hADSCs) to differentiate into pancreatic β-like cells and to examine the fate of hADSCs transplanted in STZ-NA-induced type II diabetic mice. In vitro experiments showed hADSCs could be induced to pancreatic β-like cells by three different strategies. Among them, the method combined with co-culture and differentiation medium was shown to be the most effective for the induction. In the mice model, CM-Dil dye was used to label the hADSCs for the purpose of tracking the cells. Cell transplantation was performed by tail vein injection of labeled hADSCs. By the microscopic observations on tissues sections from mice, it was found that transplanted hADSCs appeared mainly in pancreas and liver during the first month after transplantation. The labeled cells in the targeted organs were enumerated during the first two months after transplantation. The infiltration of immune cells to pancreatic tissue was decreased and the damage of the islet cells was ameliorated in the hADSCs transplanted mice compared to the sham treatment. Based on these results, it could be concluded that the hADSCs can differentiate into pancreatic β-like cells and have great potential in the application of type II diabetes treatment.

摘要
Abstract
表目錄
圖目錄
壹、研究動機與目的
貳、研究背景及重要性簡介
2.1 糖尿病
2.1.1 糖尿病之簡介
2.1.2 糖尿病的診斷
2.2 幹細胞
參、 材料與方法
3.1 人類脂肪間質幹細胞培養與繼代
3.1.1 間質幹細胞鑑定分析
3.2 人類脂肪間質幹細胞分化為類胰島 β 細胞
3.2.1 分化培養基誘導分化策略
3.2.2 人類脂肪間質幹細胞與小鼠胰臟組織共培養
3.2.3 複合式分化策略
3.2.4 萃取 total mRNA及製備 cDNA
3.2.5 聚合酶連鎖反應
3.2.6 即時定量聚合酶連鎖反應
3.2.7 Dithizone 染色
3.3 以 STZ-NA 誘發糖尿病小鼠模式探討人類脂肪間質幹細胞治療效果與追蹤
3.3.1非肥胖型第二型糖尿病小鼠模型
3.3.2 標定人類脂肪間質幹細胞與細胞移植
3.3.3 組織處理及包埋
3.3.4 常規組織染色
3.3.5 組織切片螢光拍攝前處理與免疫組織化學染色
3.4 統計分析
肆、實驗結果
4.1 幹細胞特性分析
4.1.1 幹細胞表面抗原分析
4.1.2 人類脂肪間質幹細胞分化能力
4.2 人類脂肪間質幹細胞分化為類胰島 β 細胞
4.2.1 誘導分化之細胞型態
4.2.2 誘導分化之細胞基因表現
4.2.3 誘導分化之細胞經 DTZ 染色分析
4.3 動物生理分析
4.3.1 CM-Dil 螢光染劑標定之人類脂肪間質幹細胞分化能力
4.3.2 STZ-NA 化學誘導第二型糖尿病小鼠建立及細胞移植效果
4.3.3 追蹤人類脂肪間質幹細胞
伍、討論
陸、結論與未來展望
柒、參考文獻

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網路資訊
衛生福利部,Ministry of health and welfare. http://www.mohw.gov.tw/
International Diabetes Federation, IDF. http://www.idf.org/

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