研究目的，在探討高濃度葡萄糖與抗氧化劑的維生素C、E的添加，對於培養的內皮細胞胞外間質(ECM)蛋白質，其纖維網狀蛋白Fibronectin(FN)濃度的影響，並且觀察細胞內總蛋白含量的變化，進一步觀察TGF-β與AGE是否會影響到Fibronectin的生合成。實驗方法:將105/well細胞培養於24孔well的plate中，並且分別添加M-199+10%FCS，M-199+10%FCS+30mM葡萄糖(高生理濃度)，M-199+10%FCS+5mM葡萄糖(正常生理濃度)中，維生素C(0.01mM，0.05mM)，維生素E(0.01mM，0.05mM)各別與共同添加並且又添加TGF-β與AGE抗體(0.5ug/ml)，培養72小時之後以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及以Enzyme-Linked Immunosorbance Assay(ELISA)測Fibronectin(FN)的濃度。實驗結果顯示:高濃度葡萄糖(30mM)添加比較與正常濃度葡萄糖(5mM)的添加，不管在5%或10%FCS添加於M-199培養液中，的確可使內皮細胞內Fibronectin濃度明顯的上升，而總蛋白的含量並無明顯改變，然而各別添加維生素C或E(0.01mM)的確可使存在高濃度葡萄糖的細胞內Fibronectin濃度明顯下降，但當各別添加維生素C或E(0.05mM)時，並無明顯改變高的Fibronectin生成量，而維生素C與E共同添加時二者有加乘作用皆明顯降低Fibronectin生成量，另外TGF-β與AGE抗體的確可抑制誘發Fibronectin生成。由以上結果得知內皮細胞存在高濃度葡萄糖環境下，可使ECM中Fibronectin生合成增加，並且隨著添加適當一定量的抗氧化劑維生素C、E可抑制高氧化壓力之下TGF-β誘發或氨基糖化產物- AGE (advanced glycation end product)生成所造成的Fibronectin增加，進而影響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，預防引起糖尿病血管病變(diabetic angiopathy)發生。
In this study is to explore the influence of adding high concentrated glucose and antioxidant C、E to Fibronectin(FN) a ECM protein and Quantitative changed of total cellular protein. Furthermore to examine the relationship of TGF-s and AGE to biosynthesis of FN in culture study.
Methodology of experiment: 105/well cells were incubated in 24 well plate. Each well contains M-199+10%FCS. M-199+10%FCS+30mM glucose (high physiological conc.) M-199+10%FCS+5mM glucose (normal physiological conc.) vitamin C (0.01mM.、0.05mM) and vitamin E(0.01mM、0.05mM) were added individually and in combination plus, included TGF-s and AGE antibody (0.5ug/ml) incubation were washed three times after 72hours, cell suspensions was obtained with lysis buffer. Cell lysis under Ultrasonic vibration. ELISA determinate quantitative Analysis of total cellular protein and FN concentration. The results of experiment shows that indeed the FN was significantly increase in high glucose concentration compare to normal glucose concentration Either 5%or10%FCS were present in the culture medium. Although the total quantitative of protein did not change, but adding vitamin C or E respective can cause significantly decrease of cellular FN concentration under the situation of high glucose concentration. In Addition providing vitamin C or E (0.05mM) respective were not significantly change the FN biosynthesis but add both vitamin C and E together shows additive effect on decrease fibronection biosynthesis within high glucose concentration. Furthermore TGF-s and AGE antibody can indeed reduce the Fibronectin biosynthesis.
For the data of results, we know that under the high glucose concentration can increase Fibronectin biosynthesis. Together with certain amount of antioxidant vitamin C and E can suppress the high oxidant pressures of TGF-s induction or AGE- production which cause increase fibronectin concentration production. And lead to affect the smoothness of ECM endothelium cell surface, decrease progression of cellularization of blood vessel eventually prevent the occurrence of diabetic angiopathy.
