- 學位論文
葡萄籽萃取物抑制細胞激素引起內皮細胞發炎反應的探討
Evaluation of the Inhibition Effects of Grape Seed Extract in Cytokine - Induced Endothelial Cells Inflammatory Response
摘要
發炎反應是造成許多疾病的主因。當血管內皮細胞受傷時，組織所分泌出的附著因子會增加單核球(Monocytes)對內皮細胞吸附的能力，進而移行(Migration)到內皮細胞內引發發炎反應。葡萄籽內含許多多酚化合物，根據文獻記載多酚化合物有良好的抗氧化效果。腫瘤壞死因子( Tumor Necrosis Factor-α, TNF-α )是一種常見的細胞激素，在早期的發炎反應，組織細胞會分泌TNF-α進而調控其他下游的發炎性細胞，它會刺激內皮細胞分泌黏著分子( Intercellular Adhesion Molecule-1, ICAM-1 )，造成白血球貼附在內皮細胞上。ICAM-1在正常的生理狀況時是以低濃度存在體內，但可經由細胞激素TNF-α 刺激內皮細胞而產生。 本實驗中是利用人類臍帶靜脈內皮細胞(Human Umbilical Vein Endothelial Cells, HUVECs )，在In Vitro 的狀態下以TNF-α刺激作為發炎反應模式，並添加不同濃度的葡萄籽萃取物( Grape Seed Extract, GSE)，觀察GSE是否可以保護內皮細胞，並降低細胞發炎現象。所使用的方法：內皮細胞生長( MTT Assay )、ICAM-1的表現量(Enzyme-linked immunoassay, ELISA )，人類單核球細胞株( Human monocytic leukemia cell, THP-1 )黏附至內皮細胞( Adhesion Assay )產生發炎反應的現象，及COX-2(Cyclooxygenase-2 )的表現(Western blot analysis)，最後探討GSE對內皮細胞發炎反應的調控作用。 由實驗結果得知，低濃度之GSE對於內皮細胞有促進生長的作用，但在高濃度的剌激下，會造成內皮細胞的死亡，在抗發炎的實驗中則發現，在GSE的添加與共同培養條件下，可有效的抑制TNF-α剌激內皮細胞產生發炎反應時分泌的ICAM-1含量，同時也抑制了THP-1細胞的貼附作用，此結果具有濃度的相依性。由此可推論，葡萄籽萃取物能有效抑制單核球貼附到內皮細胞上，並且可以抑制調節初期的發炎反應。
並列摘要
Inflammatory reactions usually involve complex interactions that are between circulating system, resident leukocytes and the vascular endothelium. In many studies, they demonstrated the grape seed extract (GSE) have antioxidant effect which can prevent cell injure. Therefore, we expect to study the protective effect of GSE in cytokine induced cell injure. In this study, we used the human umbilical vein endothelial cells (HUVECs) primary culture as a cell model and inducer tumor necrosis factor-alpha (TNF-α) to evaluate the anti-inflammatory functions of GSE. We also used the TNF-α (20ng/ml) to induce the inflammatory reactions in HUVECs. The cells viability, monocyte adhesion (Cell adhesion), adhesion molecular ICAM-1 (Enzyme-linked immunoassay, ELISA), cyclooxygenase-2 (COX-2) and endothelial nitric oxide synthase (eNOS) proteins expressed (western blot analysis) were detected by several assay methods in this study. The results showed that HUVECs proliferation was increased when GSE was added to the cultures. Three different concentrations of GSE (50μg/ml, 100μg/ml, and 200μg/ml) could increase cell proliferations. The GSE (50μg/ml, 100μg/ml) also showed the protective ability to reduce monocytes adhesion, and ICAM-1 secreted in inflammatory response induced by TNF-α. We found that COX-2 protein level expressed in HUVECs was induced and reduced by cultured with GSE. Our results suggested that the extract of grape seed has the possibility to be an anti-inflammatory product.