第一型糖尿病是一種自體免疫疾病，患者體內的β細胞受免疫系統攻擊而死亡，無法分泌足量的胰島素，進而使得病患失去調節血糖的能力。注射外源性胰島素是臨床常用的處置方式，然而該方法仍無法使血糖維持正常生理恆定，且造成病患生活不便。臨床研究結果顯示，胰島移植是第一型糖尿病的有效療法，可使患者本身恢復自行分泌胰島素的能力，不須依賴施打外源性胰島素來控制病情，能避免許多因糖尿病及施打胰島素而產生的併發症，並大大改善病人的生活便利性。然而目前胰島移植療法的施行率與成功率仍低，除了捐贈數量不足以外，主要原因是胰島細胞移植至肝門靜脈後，會被血液所介導的免疫反應攻擊，使得胰島細胞的存活率及功能下降。皮下組織是另一個可能的移植位置，但該區域的初始血管密度不足，無法迅速提供胰島細胞生存所需的養分及氧氣。為了解決上述問題，本論文結合臍帶血間葉幹細胞與人類臍帶靜脈內皮細胞，製備兩者均勻混合之三維幹細胞球體，希望藉其誘導血管新生及促進細胞存活等能力，提升胰島細胞移植至皮下組織後的存活率。在體外實驗中，本研究製備出三維幹細胞球體，並以免疫螢光染色、管狀形成實驗與條件培養基測試等方式證明其具有良好的誘導血管生成與促進細胞存活之潛能。動物實驗部分，我們將三維幹細胞球體與胰島素瘤細胞株MIN6細胞共同移植至小鼠皮下部位。活體冷光影像系統觀察結果顯示，與三維幹細胞球體共同移植的MIN6細胞存活情形相當良好。而由組織切片免疫染色結果可知，三維幹細胞球體可有效提升植入細胞周圍的血管新生情形，使MIN6細胞能夠在皮下部位存活並製造胰島素。由以上實驗結果可知，三維幹細胞球體可有效改善胰島細胞皮下移植後的存活率，未來或有被運用於胰島移植治療上的潛能。

Islet transplantation has been demonstrated as a promising therapy for Type 1 diabetes mellitus in addition to insulin treatment. Although offering advantages of minimally invasive operating procedure and easy access for graft monitoring, subcutaneous transplantation of islet only exhibited limited therapeutic outcomes, which can be attributable to the inadequate capability of skin tissue to foster revascularization in a short period. In this work, we fabricated three-dimensional (3D) cell spheroids composed of umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells. We hypothesize that these spheroids can induce local angiogenesis and preventing graft death to enhance the graft survival rates. According to our in vitro results, the 3D stem cell spheroids could induce neovascularization by direct formation of vessel-like structures and secretion of pro-angiogenic growth factors. Moreover, the production of pro-survival factors by the 3D stem cell spheroids was observed. The 3D stem cell spheroids and MIN6 cells were co-transplanted in subcutaneous tissue of a mouse model. Enhanced graft survival was demonstrated by tracing the bioluminescence using an in vivo imaging system. Additionally, the expression of insulin was increased significantly, as indicated by our histological analyses. These experimental data revealed the efficacy of the 3D stem cell spheroids in promoting the survival and function of subcutaneously transplanted β cells.

摘要 I
Abstract II
致謝辭 III
目錄 IV
圖目錄 VI
表目錄 VIII
第一章、緒論 1
1-1、第一型糖尿病 1
1-2、胰島細胞移植治療 2
1-3、間葉幹細胞及內皮細胞 3
1-4、甲基纖維素 5
1-5、三維細胞球體 6
1-6、研究動機與實驗目的 7
第二章、材料和方法 10
2-1、細胞培養 10
2-2、甲基纖維素水膠製備 10
2-3、三維MSC/EC細胞球體製備 11
2-4、細胞免疫螢光染色 12
2-5、管狀形成實驗 (Tube Formation Assay) 14
2-6、條件培養基(conditioned medium, CM)實驗 14
2-7、細胞存活率分析 (CCK-8 Assay) 15
2-8、細胞存活/死亡染色(Live/Dead Staining) 15
2-9、酵素連結免疫吸附分析法(ELISA) 15
2-10、注射前/後細胞球體活性觀察 16
2-11、MIN6細胞與其冷光強度線性關係測試 16
2-12、β細胞皮下移植之小鼠動物模式 17
2-13、組織病理分析與免疫螢光染色 17
第三章、實驗結果與討論 19
3-1、三維MSC/EC細胞球體之型態與特性鑑定 19
3-2、三維細胞球體血管新生因子及促生存因子表現 21
3-3、三維細胞球體促進血管新生能力 21
3-4、三維細胞球體促進β細胞存活及胰島素分泌能力 23
3-5、注射前/後細胞球體活性測試 26
3-6、MIN6細胞與其冷光強度的線性關係 27
3-7、體內細胞存活率測試 28
3-8、細胞膠體移植物巨觀型態 30
3-9、組織免疫螢光染色 31
第四章、結論 34
參考文獻 35

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