- 學位論文
胞外基質對脂肪幹細胞分化為似胰島細胞之影響
The effect of extracellular matrix on differentiation of adipose derived-stem cell into islet-like cells
摘要
第一型糖尿病是一種自體免疫疾病,會使胰島細胞凋亡而無法分泌足量的胰 島素而使病患持續性的高血糖,患者易於疲勞也增加引起酮症的機率,長期而言 也會損害周邊血管引發是網膜病變。現今只能藉由終身外源胰島素注射維持正常 的身體機能,近年多嘗試以細胞療法治療此疾病。脂肪幹細胞是一種成體幹細 胞,具有容易取得、來源豐富及具有分化複能性之特性,在醫療上極具潛力。 體內細胞的命運會受內在基因及外在微環境共同調節。微環境包含細胞間接 觸、生長因子及胞外基質 (extracellular matrix, ECM)。胞外基質是細胞所分泌的 醣蛋白,提供細胞三維結構,除此之外也會以特定的結構區域(motif)與細胞膜 上的整合蛋白 (integrin)結合,可使細胞內與細胞外進行訊息的交流,因此對幹細 胞的增生、生長及分化造成影響。 本實驗藉由研究細胞外基質對於細胞功能之影響,促使脂肪幹細胞朝向特定 方向分化。以 7-11 日齡的藍瑞斯×約克夏二品種雜交小公豬 (Sus scrofa domestica) 作為實驗動物,從其背部之皮下脂肪組織分離出豬脂肪幹細胞 (porcine adipose- derived stem cells, pADSCs),藉由本實驗室先前使用的誘導分化液將之分化成類 胰島細胞 (porcine islet-like cells, pILC)。並使用一般細胞培養皿(regular polystyrene)、幾丁聚醣修飾之細胞培養皿(chitosan-modified plate) 與幾丁聚醣 與生長因子 FGF-2 共同修飾之細胞培養皿(chitosan+FGF-2 -modified plate) 培養 與分化細胞。為去除細胞得到胞外基質之立體結構,使用弱鹼 (NH4OH) 或非離子 界面活性劑 (Triton X-100) 搭配去氧核醣核酸酶 (DNase) 溶液進行去細胞 (decellularization) 處理。以 DAPI 螢光染色檢驗細胞核之去除,使用西方點墨法 (Western blot) 檢驗細胞骨架 β-actin 以及胞外基質蛋白 fibronectin,最後以掃描式 電子顯微鏡 (Scanning electron microscope) 觀察胞外基質表面的結構。這些分析顯 示在去細胞後,細胞核及細胞內骨架大量減少,並保存胞外基質蛋白及其立體結 構。為比較 pADSC-ECM 及 pILC-ECM 的組成差異,我們分析其蛋白質組 (proteomics),並將偵測到之 7 種 ECM 蛋白以即時聚合酶鏈鎖反應 (Quantitative real time polymerase chain reaction, qPCR) 進一步確認其基因表現。最後,將豬脂 肪幹細胞重新種回 pADSC-ECM 及 pILC-ECM 以測試胞外基質是否能幫助幹細胞 分化為胰島細胞。結果顯示,胰島分化相關基因 (GLUT2, ISL1, GCK1) 之表現受 到刺激,尤其在 chitosan 或 chitosan+FGF-2 修飾之細胞培養皿上分化之細胞更為 顯著 (P<0.05)。 以上試驗結果顯示,我們建立了豬脂肪幹細胞及分化胰島細胞之去細胞方法。 並將其此外,由於胞外基質有助於胰島分化,如能更進一步的確認其中具有助益 之蛋白可有助於此後建立一個適於豬脂肪幹細胞分化為胰島素分泌細胞之微環境。
並列摘要
Type 1 diabetes (T1D) is an autoimmune disease, apoptotic pancreatic islet cells in T1D patients are unable to secrete enough insulin to control blood glucose. Hyperglycemia increases the risk of ketoacidosis, fatigue, and long-term damage of vascular retinopathy. To substitute tedious life-long exogenous insulin injections, cell therapy is a potential treatment to improve life quality of T1D patients. Among the pluripotent stem cells, the unique advantage of easily accessible, abundant and multipoint adipose-derived stem cells (ADSCs) could be a promising source for transplantation and regenerative medicine. Stem cell fate is harnessed by systemic gene expression and microenvironment (niche) which includes soluble ligands, cell communications and extracellular matrixes (ECMs) that are mostly fibril glycoproteins secreted by cells. Not only acting as supporting structures, ECMs can communicate inside and outside information by binding integrins on the cell membrane with their specific motif. This interaction is critical for the determination of the pluripotent stem-cell fate, such as self-renewal, proliferation, pluripotency and differentiation. The porcine ADSC (pADSC) was isolated from the dorsal subcutaneous fat in 7 to 11- day-old Landrace x Yorkshire hybrid piglets (Sus scrofa domestics). pADSCs were induced to porcine islet-like clusters (pILC) of insulin producing cells by chemical induction medium developed in our lab on different coating dishes with regular polystyrene, chitosan, or chitosan+FGF-2. To get the ECM structure with cell removal, decellularizing solutions such as NH4OH, Triton X-100 and DNase were combined to minimize intracellular matrix and preserve ECM protein. Levels of nucleus elimination was examed by fluorescent DAPI staining. Cytoskeletal β-actin and one of the ECM protein fibronectin were detected by western blot. Scanning electron microscope were used to observe the outlook of ECM surface. These analyses showed nuclear and cytosolic proteins were dramatically reduced and the 3-D structure of ECM were well- preserved. Proteomics were also performed to compare pADSC-ECM and pILC-ECM, seven ECM proteins were identified and individual gene expression was confirmed by real-time qPCR. Finally, pADSCs were re-seeded on pADSC-ECM or pPILC-ECM to test whether ECMs promote differentiation of islet-like cells. Expression of pancreatic islet differentiation marker (GLUT2, ISL1, GCK1) was found to be up-regulated, especially on culture dishes with chitosan or chitosan+FGF-2 (P<0.05). In conclusion, decellularizing methods for pADSC or pILC were established. Furthermore, the composition of ECMs entails pancreatic islet differentiation and identified proteins of extracellular matrix in this research may provide a suitable niche assisting pADSC differentiation into insulin-producing cells.