- 學位論文
奈米構型膠原蛋白結合Nicotinamide及Exendin-4誘導間質幹細胞成胰島素分泌細胞之研究
Study of Nano-sized Collage I Molecules combined with Nicotinamide and Exendin-4 on Differentiation of MSCs into Insulin-Secreting cells
摘要
利用菸鹼醯胺化學誘導因子可以將間質幹細胞誘導分化為β胰島細胞，這類的研究仍然著重在誘導劑的應用，缺少進一步探討細胞外基質對間質幹細胞誘導成β胰島細胞之影響。亦有不少研究單純以模仿及製造細胞外基質生長環境來進行對間質幹細胞分化之影響，但卻甚少把細胞外基質微小化並探討對於細胞之相互作用。本研究的動機與目的就是發展出不同膠原蛋白奈米構形及結合Nicotinamide、Exendin-4化學誘導因子來誘導間質幹細胞成胰島素分泌細胞之研究。實驗中將膠原蛋白製備成奈米級生物分子之微粒形與纖維形，使細胞膜上受體與細胞外基質分子吸附之探討，並結合Nicotinamide及Exendin-4將之應用在間質幹細胞誘導成胰島素分泌細胞之培養上。從細胞結果可以看出，間質幹細胞在培養後可觀察到間質幹細胞型態逐漸誘導成葡萄串型態之胰島細胞。且加入第一型膠原蛋白奈米級纖維後，間質幹細胞誘導成胰島細胞的數目比單純之Nicotinamide及Exendin-4誘導多且明顯。顯示第一型膠原蛋白奈米級分子是有促進間質幹細胞分化之效果。在免疫螢光結果顯示，間質幹細胞經培養至14天後，方可觀察到紅色之insulin蛋白表現，隨著培養天數增加，insulin表現量明顯增加。經由上述之結果分析所得：第一型膠原蛋白奈米級分子是有促進間質幹細胞分化成胰島細胞之效果。於動物實驗的結果，利用高脂飼料餵食大鼠，並於腹腔注射STZ後，經過葡萄糖耐受性測試、葡萄糖-胰島素耐受性測試及糖化血色素指數之後，確定可以誘導出第二型糖尿病鼠，並將誘導完成之胰島素分泌細胞移植於胰臟，並進行蘇木-伊紅染色觀察。並建立動物胰島缺陷實驗模型，再以所培養之間質幹細胞體外誘導成之胰島素分泌細胞進行植入至胰臟中，評估其胰島素分泌調節之功能。
並列摘要
Recent research reports indicated that MSCs could be differentiated into Insulin-secreting cells by Nicotinamide. However, these studies focused on the usage of pure Nicotinamide chemicals and lacked details regarding the influence/contribution of ECM in combination with induction chemicals. Also many related researches focused on the preparing bio-mimetic scaffolds very limited imevaluated the influence of nano-sized order ECM on the cellular interactions. The motivation and purpose of this study are to develop a different configuration of the collagen, and combine the induction chemicals, Nicotinamide and Exendin-4 with the differentiation of MSCs into insulin secreting cells. The adhesion and interactions between the cellular receptor and nano-sized collagen molecules, and the probably contribution of these molecules on the differentiations of MSCs into insulin secreting cells will also be included. Established animal experimental model of islet defect, then between mesenchymal stem cells cultured in vitro induction of insulin secretion cells implanted into the pancreas conducted to assess the regulation of insulin secretion function. Preparation collagen form of fibers and nanoparticls biological molecules, explore receptor extracellular matrix molecules adsorbed on the cell membrane, and combined Nicotinamide and Exendin-4 will be the application of mesenchymal stem cells into insulin secreting cells cultured. From the cells results, can be observed after culture mesenchymal stem cell morphology gradually induced into bunches type of islet cells. After adding the type I collagen and nano-fibers, mesenchymal stem cells into islet cells than simply the Nicotinamide and Exendin-4 multi-inducing and obvious. Display type I collagen molecule is effective promote differentiation between mesenchymal stem cells. Results are shown in immunofluorescence, mesenchymal stem cells were cultured for 14 days before observation of the red protein expression of insulin, significantly increased expression of insulin. Analysis of the results type I collagen molecules are nanoscale between promoting effects of mesenchymal stem cells to differentiate into islet cells. On the results of animal experiments, rats fed high-fat diet, and after intraperitoneal injection of STZ, after a glucose tolerance test, glucose - insulin tolerance test and glycosylated hemoglobin index after determining a second type can be induced diabetic rats and completion of the insulin-secreting cells induced transplanted pancreas, and hematoxylin - eosin staining. And the establishment of animal experimental model of islet defect, then between mesenchymal stem cells cultured in vitro induction of insulin-secreting cells were implanted into the pancreas, to assess the function of regulating insulin secretion.