- 學位論文
結合螢光與基因工程以建立可偵測GLUT4轉運及葡萄糖攝取能力之細胞平台
A combination of fluorescence detection and genetic engineering the establishment of a cell platform to evaluate GLUT4 translocation and glucose uptake
摘要
隨著現代飲食習慣的改變，糖尿病為全球性之健康問題，並有年輕化的趨勢。血液中的葡萄糖主要由骨骼肌細胞與脂肪細胞攝取和分解，葡萄糖轉運蛋白4 (glucose transporter 4, GLUT4) 對細胞在血糖升高時所誘導之攝取作用中扮演重要角色，其功能受胰島素所調控，當體內出現胰島素阻抗時，降低細胞對胰島素刺激的靈敏度，使細胞無法有效攝入葡萄糖，造成高血糖的現象。然而目前檢測葡萄糖攝取的方法除了價格昂貴、耗時、步驟繁瑣外，在檢測原理上也容易受細胞內其它代謝途徑影響，或是不能與生理環境比較。基於上述缺點，我們希望能提供一個快速、直接的檢測平台，以完善整個葡萄糖攝取的分析流程。因此本研究以GLUT4作為標的，轉染可表現其重組蛋白和綠色螢光蛋白— eGFP的質體到CHO-K1細胞，隨後利用抗生素篩選出能穩定及大量表現此重組蛋白之細胞株，透過分析螢光重組蛋白的螢光強度與分佈，確認殖株的表現量，以及在胰島素處理下偵測到螢光的轉位，顯示此GLUT4重組蛋白對胰島素具有反應。後續利用全內反射螢光顯微鏡偵測細胞膜上的螢光數量，並與傳統檢測法進行確效與比較，顯示此檢測方法的結果與葡萄糖的攝取具有正相關性，代表此細胞平台可應用於日後篩選具促進葡萄糖攝取潛力的樣品。
並列摘要
Diabetes becomes a global health issue as a result of the shifting of dietary habits. Previously, the population of patients was seen mainly in the middle-aged and elderly, but it is getting more frequently among young people. Glucose serves as energy source for most cells. Blood sugar rises after a meal and is mainly absorbed by skeletal muscles and adipose tissues. Glucose transporter 4 (GLUT4), the downstream effector of insulin, takes an important role in glucose uptake. Insulin resistance would cause defective insulin response in target cells, leading to hyperglycemia. However, approaches in measuring glucose uptake are considered to be time consuming, expensive and not easy to operate. Also, the results may be affected by other metabolic pathways in cells, and is incompatible with the actual physiological environments. According to these disadvantages, we attempted to establish a more rapid and intuitive method to measure glucose uptake through the construction of a cell platform. Based on this reason, we transfected a recombinant plasmid into CHO-K1 to produce GLUT4-eGFP recombinant protein. Antibiotics selection was carried out to exclude the non-transfected cells. The fluorescence intensity was analyzed to estimate the stability and expression efficiency of this fusion protein. In addition, the GLUT4-eGFP recombinant protein is responsive upon insulin stimulation, and the fluorescence was detected by Total internal reflection fluorescent microscope (TIRFM) afterwards. The result shows that our detection method is correlated to glucose uptake and is more efficient than the traditional methods, indicating that this cell platform can be applied to screen potential compounds with the ability to increase cellular glucose uptake.