添加維生素C與尿酸於高葡萄糖壓力下對細胞之纖維網狀蛋白增生影響之探討

糖尿病是近幾年來罹病率逐年增高的慢性病，長時期的高血糖會使血液中自由基增加，造成內皮細胞的損傷，進而導致多種併發症的產生。將人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cell；HUVEC)與狗的腎臟上皮細胞(Madin-Darby Canine Kidney；MDCK)培養於不同的葡萄糖濃度下，觀察纖維網狀蛋白、細胞存活、過氧化氫、麩胱苷肽過氧化酶的影響，與血管內皮生長因子(Vascular Endothelial Growth Factor：VEGF)的相互關係；再藉由添加抗氧化劑，觀察上述變化的情形，此外也另外添加糖化終產物抗體(Advanced Glycosylation End Products；AGE)，看上述現象是否與糖化終產物有關。方法為將細胞培養於不同葡萄糖濃度的培養液中，再個別或共同添加維生素C與尿酸培養72小時後收集細胞，利用ELISA方式測定纖維網狀蛋白(Fibronectin ；FN)；Horseradish Peroxidase (HRPase)檢測系統測定過氧化氫；分光光度計測定麩胱苷肽過氧化酶的活性；採用分析乳酸去氫酶方式間接觀察細胞死亡的情形；西方墨點法確認VEGF。結果顯示：隨著葡萄糖濃度的增加會使細胞的纖維網狀蛋白與過氧化氫含量增加，麩胱苷肽過氧化酶活性降低，VEGF含量增加；再添加抗氧化物質後，細胞的纖維網狀蛋白與過氧化氫濃度則下降，麩胱苷肽過氧化酶活性有上升傾向，只有添維生素C時，VEGF含量才會下降；添加AGE抗體後，效果更為顯著，證實細胞培養在高葡萄糖環境下，確實會產生AGE，且上述檢測的物質，都與AGE的形成相關。依上述結果得知：糖尿病病患長期處於高血糖下會使自由基含量升高，纖維網狀蛋白含量增加，VEGF增高，進而帶動血管增生，造成血管性病變。當補充適量的維生素C，或血液中有適量的尿酸時，則可能可以延緩糖尿病血管方面的併發症。

The patients with chronic diseases, particularly Diabetes Mellitus increased year by year. If blood sugar is continuous over the normal level for a long time, the free radical level is increased and causes significant damages in endothelial cells. This further enhances the complications of Diabetes Mellitus. In this study, human umbilical vein endothelial cell (HUVEC) and Madin-Darby canine kidney (MDCK) were cultured under different concentrations of glucose. And the relationship among fibronectin (FN), H2O2, glutathione peroxidase (GPx), and the survivors of the cells upon vascular endothelial growth factor (VEGF) were examined. Also, the effect of advanced glycosylation end products (AGE) in the cells cultured with ascorbic acid or/and uric acid was assayed. The following methods such as ELISA, horseradish peroxidase (HRPase) test system, UV/VIS spectrophotometer, and Western blotting were used. The results revealed that high glucose level led to significant increase in VEGF content, and the level of fibronectin and H2O2, and GPx level reduced. Furthermore, we proved the addition of AGE antibody in cultured cells with high glucose concentration significantly affected the above observations. The DM patients with high level of free radicals resulted in proliferation of blood vessels and angiopathy (ex: atherosclerosis, angiogenesis, etc.), this likely due to the increases of fibronectin and VEGF. And when the moderate ascorbic acid or/and uric acid in DM patients may delay the development of complications in blood vessels.