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研究生:盧盈叡
研究生(外文):Lu, Ying-Jui
論文名稱:探討人類臍帶間質幹細胞移植至NOD小鼠後所產生之免疫抑制的效果
論文名稱(外文):Immunosuppressive Effects Of Human Umbilical Cord Stromal Mesenchymal Stem Cells Transplantation In NOD Mice
指導教授:徐佳福林谷峻林谷峻引用關係
指導教授(外文):Shyu, Jia-FwuLin, Gu-Jiun
口試委員:徐佳福陳天華林谷峻
口試委員(外文):Shyu, Jia-FwuChen,Tien-HuaLin, Gu-Jiun
口試日期:2012-05-31
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:67
中文關鍵詞:人類臍帶間質幹細胞瓦頓氏膠間葉幹細胞β細胞非肥胖型糖尿病小鼠免疫調節第一型糖尿病
外文關鍵詞:human umbilical cord stromal mesenchymal stem cellsWJ-MSCNOD mouseWharton’s Jelly-mesenchymal stem cellsImmunomodulatory
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第一型糖尿病是種自體免疫疾病,因為T細胞攻擊β細胞,造成胰島素分泌量不足。而來自於人類臍帶的間葉幹細胞具有免疫抑制的能力,可作為再生醫學的工具。我們的研究目的是研究非肥胖糖尿病小鼠經由眼窩注射用慢病毒感染綠螢光蛋白的間葉幹細胞後其免疫抑制效果。表現綠色螢光的間葉幹細胞在移植非肥胖糖尿病小鼠後,會降低血糖值,增加存活率及較少的嚴重性淋巴球浸潤現象。而人類 C-peptide與綠色螢光共同表現在同一個細胞上,表示間葉幹細胞會分化成胰島素生產細胞。在移植人類臍帶間葉幹細胞後,在小鼠脾臟中發現會Th2和Th17的表現降低,而調節性T細胞和產生IL-10的CD4+ T細胞表現增加。在胰臟淋巴結中觀察到樹突細胞表現降低的現象。經CFSE稀釋分析,發現間葉幹細胞移植後會CD4+ T細胞的增生能力降低。總之,MSC在體外和體內實驗都具有分化成胰島素生產細胞的能力。間葉幹細胞抑制Th2和Th17細胞的反應,促進調節性T細胞和IL-10的CD4+ T細胞的增生。間葉幹細胞會影響樹突細胞的抗原呈現及分化能力。間葉幹細胞能降低CD4+ T細胞的增殖。移植表現綠螢光蛋白的人類臍帶間葉幹細胞,會抑制非肥胖糖尿病小鼠的第一型糖尿病復發,還可利用GFP進行間葉幹細胞的追蹤。
Type 1 diabetes is caused by T cell mediated autoimmune destruction of pancreatic β-cells. Mesenchymal stem cells (MSCs) derived from Wharton ’ s jelly of human umbilical cord may represent a more promising regenerative medicine tool that have immunosuppressive effects. The purpose of this study was to study the therapeutic effect of MSCs in non-obese diabetic (NOD) mice by orbital injection of lentivirus-transduced MSCs. GFP-labeled MSCs injection leaded to significant decreases of blood glucose, higher survival rate and less severe insulitis in NOD mice. Colocalization of human C-peptide and GFP in pancreas implies that MCSs differentiation to insulin-producing cells. Transplantation of transduced MSCs decreased the Th2 and Th17; increased CD4+CD25+FoxP3+ regulatory T cells (Treg) and IL10 producing T cells population of CD4+ T cells in the spleen of NOD mice. In the PLN, MSCs transplantation decreased dendritic cells (DC) population. CFSE dilution assay showed that MSCs transplantation attenuated CD4+ T cell proliferation. In conclusion, MSC possess the ability, both in vitro and in vivo, to differentiate into insulin-producing cells. MSCs suppress Th2 and Th17 responses by promoting Treg and IL10 producing T cells population. MSCs had effects DC antigen presentation and differentiation. MSCs treatment reduced the proliferation capacity of CD4+ T cell. The transplanted MSCs could be tracked by GFP and suppresses autoimmune recurrence in NOD mice.
正文目錄…………………………………………………………………I
圖目錄……………………………………………………………………V
中文摘要………………………………………………………………VI
英文摘要………………………………………………………………VII
第一章 緒言……………………………………………………………1
第一節 第一型糖尿病……………………………………………1
壹、病因…………………………………………………………1
貳、致病機制……………………………………………………2
參、症狀與併發症……………………………………………3
肆、治療策略……………………………………………………3
第二節 間葉幹細胞………………………………………………4
壹、間葉幹細胞與再生醫學……………………………………4
貳、人類臍帶瓦頓氏膠間葉幹胞………………………………6
第三節 免疫調控…………………………………………………7
壹、免疫系統…………………………………………………7
一、先天免疫系統…………………………………………7
二、後天免疫系統…………………………………………8
貳、輔助型T細胞與第一型糖尿病的關聯性…………………9
一、第一型輔助T細胞和第二型輔助T細胞……………9
二、第十七型輔助T細胞…………………………………10
三、調控型T細胞…………………………………………10
參、調控型T細胞……………………………………………11
肆、介白素-10…………………………………………………12
第四節 基因轉殖………………………………………………12
壹、非病毒載體………………………………………………13
貳、病毒載體…………………………………………………13
參、慢病毒載體………………………………………………14
第五節 第一型糖尿病動物模式………………………………15
壹、藥物誘導糖尿病動物模式………………………………15
貳、自發性糖尿病動物模式…………………………………16
第六節 研究動機與目的………………………………………17
第二章 材料與方法…………………………………………………19
第一節 實驗材料………………………………………………19
壹、實驗藥品,試劑與耗材…………………………………19
貳、實驗基因,細胞,菌種及載體…………………………25
第二節 實驗方法………………………………………………26
壹、體外培養細胞與基因轉導………………………………26
一、293FT 細胞株及間葉幹細胞的培養…………………26
二、綠螢光蛋白與慢病毒質體的製備……………………26
三、基因轉染(Transfection)…………………………28
四、收集病毒………………………………………………28
五、感染間葉幹細胞(Transduction)…………………29
貳、動物實驗…………………………………………………29
一、眼窩注射移植幹細胞(Transplantation)………29
二、血糖數值偵測…………………………………………29
三、眼窩採血法……………………………………………30
四、犧牲取臟器……………………………………………30
叁、免疫細胞分析……………………………………………30
一、犧牲取臟器……………………………………………30
二、淋巴細胞實驗前的前置處理…………………………31
三、細胞表面抗原與細胞內細胞因子染色………………32
四、淋巴細胞增生染色……………………………………33
肆、免疫組織化學染色………………………………………34
第三章 結果…………………………………………………………35
第四章 討論…………………………………………………………41
第五章 結論…………………………………………………………48
第六章 參考文獻……………………………………………………50
第七章 結果圖表……………………………………………………61

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