臺灣博碩士論文加值系統

目前糖尿病（DM）已成為二十一世紀全世界最重要的公共衛生課題之一，隨著病患人數的劇增，糖尿病威脅著全人類的健康。而第一型糖尿病（T1D）的病因為自體免疫攻擊胰島細胞，T1D的病患佔總糖尿病的病患5~10%。注射胰島素治療T1D，並無法阻止自體免疫攻擊胰島細胞。隨著再生醫學的發展，目前提出了幾種可能有治癒T1D潛力的治療方法，其中幹細胞療法特別有潛力。根據我們實驗室之前研究的結果，瓦頓氏凝膠間質幹細胞（WJ-MSCs）可以降低非肥胖型糖尿病（NOD）小鼠的高血糖。而WJ-MSCs在NOD小鼠體內可以調控免疫反應以及分化成胰島素分泌細胞（IPCs)。但是NOD小鼠的血糖並沒有恢復到正常範圍。在這個研究中，我們藉由轉導pancreatic and duodenal homeobox 1 (PDX1)來誘導WJ-MSCs分化成為IPCs。為了瞭解WJ-MSCs歸巢的機制，我們通過慢病毒引入熒光素酶及綠螢光蛋白接著使用活體影像分析儀（IVIS）去追踪IPCs。此外，我們建立了刺激WJ-MSCs分化變成IPCs以及活體追踪這些細胞的方法。另外，我們還計劃引入更多的潛在基因如PAX4，NKX6.1能夠更有效地誘導WJ-MSC分化成IPCs。最後，幹細胞的增殖和後續的分化，似乎具有相當大的潛力，以克服器官捐贈的短缺。細胞替代療法可能會提供最好的方式來實現對糖尿病患者生理上的血糖控制。

Diabetes mellitus (DM) threatens people health all over the world. Type 1 diabetes mellitus (T1D), caused by autoimmune attacking islet cells, affects 5-10% of total DM. Insulin injection is the predominant treating of T1D that cannot prevent islet cells loosing and poor immunosuppression. The recent progress in regenerative medicine, especially stem cell therapy, has suggested several novel and potential cures for T1D. According to our previous study, Wharton’s jelly mesenchymal stem cells (WJ-MSCs) can reduce hyperglycemia in nonobesity diabetic (NOD) mice. The function of WJ-MSCs is probably due to immune-modulatory and they may become insulin producing cells (IPCs) in vivo. However, blood sugar does not return to normal rang in NOD mice. In this study, we induced WJ-MSCs into IPCs by transduction of pancreatic and duodenal homeobox 1 (PDX1) in these cells. To reveal the mechanism of WJ-MSCs homing, we introduce luciferase and GFP by lentivirus and use IVIS Spectrum to track IPCs in vivo. In addition, we establish a method of stimulating MSCs to IPCs differentiation and tracking these cells in vivo. Furthermore, we plan to introduce more potential genes like PAX4, NKX6.1 to drive WJ-MSC to IPCs more effectively. In conclusion, the expansion and subsequent differentiation of stem cells, appears to have considerable potential to overcome the shortage of donor organs. Cellular replacement therapy may offer the best approach to achieve physiologic glucose control in diabetic patients.

正文目錄 VII
圖表目錄 X
中文摘要 IV
英文摘要 V
第一章 緒論 6
　第一節 糖尿病（Diabetes mellitus） 6
壹、病因 6
貳、併發症 7
參、分類 7
肆、診斷 9
伍、治療 9
陸、預防 10
　第二節 間葉幹細胞（Mesenchymal stem cell） 11
壹、幹細胞的分類 11
貳、幹細胞產生新的胰島β細胞的發展 12
參、瓦頓氏凝膠間葉幹細胞（Wharton’s jelly mesenchymal stem cells） 13
第三節 基因轉殖 14
　　壹、病毒載體 15
貳、非病毒載體 18
　　參、基因 21
第二章 實驗材料與方法 23
　第一節 實驗材料 23
　　壹、實驗藥品、試劑與耗材 23
　　貳、實驗基因、細胞、菌種及載體 26
　　參、實驗儀器設備 26
　第二節　實驗方法 27
　　壹、WJ-MSCs的培養 27
　　貳、293FT細胞株的培養 27
　　參、慢病毒質體製備 27
　　肆、基因轉染（Transfection） 28
　　伍、收集病毒 29
　　柒、慢病毒感染WJ-MSCs（Transduction） 30
　　捌、體外生物發光檢測（In vitro bioluminescent assay） 30
　　玖、腺病毒（Adenovirus）放大以及感染 30
　　拾、腺病毒（Adenovirus）感染WJ-MSCs 31
　　壹拾壹、細胞免疫螢光染色 31
第三章 結果 32
　第一節 慢病毒質體製備 32
　第二節 限制酶酶切（Restriction digestion） 32
　第三節 基因轉染（Transfection） 33
　第四節 慢病毒試紙檢測 33
　第五節 慢病毒感染WJ-MSCs 33
　第六節 體外生物發光試驗 34
　第七節 使用螢光顯微鏡檢查體外生物發光試驗 34
　第八節 WJ-MSCs免疫螢光染色 35
第四章 討論 36
第五章 參考文獻 40
第六章 實驗圖表 43
第七章 附錄 51

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