脂肪幹細胞 (ADSCs) 具有調控與分泌多種脂肪激素的功能，因此在治療第二型糖尿病上具有巨大的潛力。脂肪細胞所分泌的脂聯素是已知可以改善代謝異常、免疫發炎反應以及胰島素阻抗性的抗糖尿病脂肪激素。本研究以脂聯素為標靶，篩選可提高 ADSC 脂聯素表現之藥物，並探討高脂聯素表現之 ADSCs 應用於第二型糖尿病治療之可行性以及其作用機制。研究中以 PT56 ADSCs 為藥物篩選平台，我們發現小分子藥物 TW03 可以藉由降低 Adipo-R2 中的 DNA 甲基化狀態，增加 ADSCs 的脂聯素表現量。研究中利用高脂飼料 (HFD) 誘發第二型糖尿病小鼠模型，評估 TW03 預處理的 ADSC (即高脂聯素表達之 ADSCs) 移植後的療效。相較於單獨 ADSC 移植，我們發現 TW03 預處理的 ADSC 移植更能顯著地改善肥胖與胰島素阻抗性相關的主要參數，包含空腹血糖值、胰島素值、三酸甘油脂、總膽固醇濃度、葡萄糖耐受性以及胰島素阻抗性指標。這些療效可能是來自 ADSCs 的脂聯素，經由旁分泌作用激活肝臟細胞中的 AMPK 和 P38 MAPK 途徑。研究中也發現 TW03 預處理的 ADSC 移植後可以降低肝臟中 NF-κB 的活化程度以及 NF-κB 所轉錄的目標基因 IL-1β 和 TNF-α 的表現量，而這些細胞保護作用促進第二型糖尿病鼠肝臟葡萄糖代謝的修復。對於第二型糖尿病的療效，本研究結合了小分子藥物與 ADSC 移植，提供了可能的治療機制以及新的治療策略。

Adipose-derived stem cells (ADSCs) show great clinical potential on treating type 2 diabetes due to their abilities for regulation/secretion of adipose-associated cytokines. Adiponectin is an anti-diabetic adipokine secreted by adipocytes and has been found related to metabolic dysfunctions, inflammation and insulin resistance. Using adiponectin as the target, the aim of this study is to screen for drugs that increase the expression of adiponectin in ADSCs, and to investigate the feasibility and mechanism of ADSCs with high adiponectin expression in the treatment of type 2 diabetes. In this study, using PT56 ADSCs as a drug-screening platform, we found that the small molecule TW03 could increase the expression level of adiponectin in ADSCs by reducing DNA methylation statuses in Adipo-R2. The therapeutic efficacies of TW03-pretreated ADSCs’ transplantation were evaluated using high fat diet (HFD)-induced type 2 diabetic mouse model. In comparison with the untreated ADSCs, it was found that the transplantation of TW03-pretreated ADSCs led to the improvement in key parameters related to obesity and insulin resistance, such as fasting blood glucose, insulin, triglyceride, total cholesterol levels, IPGTT and HOMA-IR values. The therapeutic benefits might be attributed to the high amount of adiponectin which activated AMPK and P38 MAPK pathways in hepatocytes via paracrine signaling. It was also demonstrated that the NF-κB activation status was reduced and transcriptional targets of NF-κB (i.e. proinflammatory cytokines including IL-1β and TNF-α) were also decreased due to the TW03-pretreated ADSCs’ transplantation. These cytoprotective effects resulted in the restoration of hepatic glucose metabolism in type 2 diabetic mice. This study provides putative therapeutic mechanisms and a novel strategy including a small molecule drug combined with ADSC transplantation that delivers the therapeutic benefits for type 2 diabetes.

中文摘要…………………………………………………………………………...…………......................I
英文摘要……………………………………………………………………………...........................III
目錄……………………………………………………………………………………..........................………V
一、 研究動機與研究目的………………………………………………………………..............……1
二、 背景及重要性介紹……………………………………………………………...................3
2.1 糖尿病……………………………………………………………………………….........................3
2.2 脂肪幹細胞……………………………………...……………………………….....................…4
2.3 幹細胞在第二型糖尿病治療的應用現況…...………….……………….........………….4
2.4 脂聯素…………………………………….………………………………………….......................8
2.5 高油脂飼料誘導第二型糖尿病………………………………………………..............……..9
三、 研究材料與方法…………………………………………………………................……………11
3.1 實驗設計……………………………………………………………………….......................……11
3.2 人類脂肪幹細胞來源及培養條件...…………………………………............…………….12
3.3 實驗小鼠飼養……………………………………………………………….....................………12
3.4 糖尿病鼠的誘導和脂肪幹細胞移植..…………………………………………….............12
3.5 空腹葡萄糖耐受性試驗……………………………………………………...…...............….13
3.6 酵素連結免疫分析法……………………………...……………………………................….13
3.7 血糖、總膽固醇和三酸甘油脂測定……………………………...…...........…………….13
3.8 西方轉印法……………………………………………………………………….......................14
3.9 即時聚合酶連鎖反應…………………………………..……………………….................….14
3.10 亞硫酸鹽定序……………………………..…………………………………….....................15
3.11 蘇木精–伊紅染色………………………………………………………….................…...….16
3.12 統計分析………………………………………………………………….………......................16
四、 實驗結果與討論……………………………………………………………………...............….17
4.1 小分子藥物 TW03 能提升 PT56 脂肪幹細胞脂聯素基因表現，且具有濃度
依賴和時間依賴性…………………………………….…………….....................……….…17
4.2 小分子藥物 TW03 可以藉由降低 Adipo-R2 甲基化程度，進而提升 PT56
脂肪幹細胞脂聯素基因表現……………………………………………….................……….19
4.3 高油脂飼料誘導二型糖尿病模型確認…………………….………………...........…..….22
4.4 TW03 藥物預處理之 PT56 脂肪幹細胞移植後能改善血糖、血脂、葡萄糖耐
受性和二型尿病相關血液生化數值…..……………………………………............……...23
4.5 TW03 藥物預處理之 PT56 脂肪幹細胞移植可藉由調控糖尿病鼠肝臟中的粒
線體生成、糖解和糖質新生作用相關酵素基因表現來改善葡萄糖代謝…...….…30
4.6 TW03 藥物預處理之 PT56 脂肪幹細胞移植後能增加糖尿病鼠肝臟中AMPK
和 p38 MAPK 的活化，並在短期內降低肝臟的免疫發炎反應…………….....…..33
五、 實驗結論………………………………………………………………….………................….….41
六、 參考文獻………………………………………………………………………………...................43

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