維生素C、E和尿酸對高葡萄糖情況下內皮細胞胞外間質蛋白和一氧化氮之影響

糖尿病是長期處於高葡萄糖的環境下，伴隨著有抗氧化系統失衡和自由基增加的情形發生。抗氧化維生素C、E和尿酸被提及具有抗氧化的特性。因次本實驗的研究目的在探討高濃度葡萄糖的環境下，添加維生素C、E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。藉由添加AGE抗體來觀察上述之影響是否由糖尿病所產生的氧化產物AGEs所造成。實驗方法是將人類臍靜脈內皮細胞(HUVEC)培養於不同葡萄糖濃度(0、5、10、30mM)、維生素C(0.05mM)、維生素E(0.05mM)、尿酸(3、5、10mg/dl)下，另也添加AGE抗體(0.5μg/ml)，培養72小時後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：隨著葡萄糖濃度上升，纖維網狀蛋白含量增加、細胞死亡情形也隨之上升。添加維生素C、E和尿酸則會降低纖維網狀蛋白、NO和延緩細胞死亡。另外，AGEs產生的確會引起纖維網狀蛋白增加，但與NO和細胞死亡無關，因此由上述結果得知，內皮細胞存在高濃度葡萄糖環境下，可使細胞外間質蛋白─Fibronectin生合成增加。藉由添加抗氧化劑維生素C、E和尿酸，的確抑制了高氧化壓力下AGE生成所造成的纖維網狀蛋白含量增加，也使自由基的產量下降和延緩細胞死亡的情形，進一步影響了內皮細胞血管腔表面平滑程度，防止血管內纖維增生的進行，而預防了糖尿病所造成的血管病變。

Diabetes mellitus is a condition under high glucose environment accompanied with the unbalance of antioxidative systems and the elevation of free radical. It has been reported that vitamin C, E and uric acid seem to be antioxidants. Therefore, the purpose of this study is to investigate the influence on ECM, level of nitric oxide, and cell death with the addition of vitamin C, E and uric acid in a high-concentration glucose environment. By adding AGE antibody, it was possible to decide whether the fore-mentioned effects caused by AGEs, the oxidized products of diabetes mellitus. The human umbilical vein endothelial cells ( HUVEC ) were incubated in glucose ( 0, 5, 10, 30mM ), vitamin C ( 0.05mM ), vitamin E ( 0.05mM ), uric acid ( 3, 5, 10mg/dl ) and AGE antibody ( 0.5µg/ml ) for 72 hours. Then the fibronectin ( FN ), nitric oxide ( NO ) and cell death were measured by ELISA, NO kit and lactate dehydrogenase ( LDH ) kit respectively. The results of experiment showed the FN and cell death ( LDH release ) increased significantly in high glucose concentration. And FN, NO content and cell death were decreased with the addition of vitamin C, E and uric acid. Also, AGEs would increase FN biosynthesis, but had nothing to do with NO content and cell death. These results suggested that FN biosynthesis increased in high glucose concentration. A antioxidants vitamin C, E and uric acid could suppress the formation of AGEs which resulted in the increase of fibronectin, the decrease of free radical, and the deferment of cell death under high oxidant stress. Moreover, it would lead to the smoothness of ECM endothelium cell surface to decrease progression of cellularization of blood vessel, which would eventually prevent the occurrence of blood vessel disease caused by diabetic mellitus.