| Abstract: | 糖尿病是一常見的代謝疾病。長期高血糖會使反應性氧化物質(ROS)產生，除了會使血管內皮細胞損傷外，也是造成許多併發症的發生原因。維生素C和尿酸在體內有抗氧化劑的角色。本實驗是主要探討培養在高葡萄環境下，添加維生素C與尿酸，對於內皮細胞胞外間質蛋白(ECM)、一氧化氮含量和細胞死亡的影響。實驗方法是將人類臍靜脈培養於不同濃度的葡萄糖(0、5、10、30 mM)中，維生素C(0.05mM)、尿酸(0、3、5、10mg/dl)各別或共同添加，另也添加糖化終產物抗體(AGEs antibody, 0.5mg/ml)，看糖化終產物與上述現象是否有關。培養七十二小時後，以PBS清洗三次，利用Lysis buffer使細胞懸浮，超音波振盪破壞細胞，分析細胞內總蛋白含量、以酵素接合免疫吸附法(ELISA)、NO Kit、LDH Kit分析各組細胞內纖維網狀蛋白(Fibronectin)、NO含量和細胞死亡的情形。結果顯示：隨著葡萄糖濃度增加使內皮細胞中Fibronectin濃度明顯上升，細胞死亡情形也增加，NO則減少。添加維生素C及尿酸會降低纖維網狀蛋白，減少細胞死亡的情形。另外，將尿酸與維生素C共同添加則更明顯抑制纖維網狀蛋白的合成。AGEs抗體添加的確可抑制誘發Fibronectin生合成增加。由以上結果得知，內皮細胞在高濃度葡萄糖環境下，可使ECM中Fibronectin生合成增加，並且隨著添加維生素C和尿酸等抗氧化劑，可抑制高糖氧化壓力下生成糖化終產物(AGEs)所造成的Fibronectin含量增加，進而避免內皮細胞功能失調，預防引起糖尿病血管病變的發生。
Diabetes mellitus (DM) is the most common metabolic disease. Prolong hyperglycemia induces reactive oxygen species in the body, results in endotheial cell injury, and enhances the development of diabetic complications. Ascorbic acid and uric acid play the rules of antioxidants in our body. Therefore, this study investigates the influence on extracellular matrix (ECM), level of nitric oxide (NO), and cell death with the addition of ascorbic acid and uric acid in a high glucose concentration environment. In the other hand, by adding advanced glycation end products (AGEs) antibody, it was possible to determine that the afore mentioned effects whether caused by the formation of AGEs. The human umbilical vein endothelial cells (HUVEC) were incubated with glucose (0,5,10,30 mM), ascorbic acid (0.05 mM), uric acid (3,5,10 mg/dl) and AGEs antibody (0.5mg/ml) for 72 hours. Then wash three times with Phosphate buffered saline (PBS), cell suspensions were obtained with lysis buffer. Cells were lysed under ultrasonic vibration. The level of fibronectin (FN), and NO, and cell death were measured by enzyme-linked immunosorbent assay (ELISA), NO kit and lactate dehydrogenase (LDH) kit, respectively. Results showed the FN and cell death (LDH release) increased significantly in a high glucose concentration. And FN, NO content and cell death were decreased with the addition of vitamin C and uric acid. A combination of vitamin C and uric acid exhibits a significant synthesis of fibronectin. Also, AGEs antibody would decrease FN biosynthesis. These results suggested that FN biosynthesis increased in high glucose concentration. An antioxidants vitamin C and uric acid could suppress the formation of AGEs what resulted in the increase of fibronectin, the decrease of free radical, and the deferment of cell death under high oxidant stress. Moreover, it would avoid the endothelial dysfunction which would eventually prevent the occurrence of blood vessel disease caused by DM. |
