- 學位論文
表沒食子兒茶素沒食子酸酯抑制甲基乙二醛誘導骨髓間葉幹細胞產生氧化壓力之探討
Epigallocatechin-3-gallate Prevents Methylglyoxal-induced Oxidative Stress in Bone Marrow Mesenchymal Stem Cells
摘要
糖尿病患者血漿中含有高含量的甲基乙二醛 (MG)，此活性雙羰基物質會與 DNA 與蛋白質形成不可逆的糖化作用終產物 (AGEs)，造成細胞損傷；因此，MG 在糖尿病併發症上扮演重要的角色。表沒食子兒茶素沒食子酸酯 (EGCG) 是兒茶素中含量最多的一種，具有多酚類的結構，能清除許多種類的自由基，並能有效的捕捉 MG，抑制 AGEs 的形成。本研究旨在探討 MG 是否會使人類骨髓間葉幹細胞 (hBMSCs) 產生氧化壓力，進而使細胞走向凋亡，並觀察 EGCG 是否能夠抑制 MG 的作用。首先分析不同濃度 MG 或 EGCG 個別對 hBMSCs 活性之影響；再將 MG、EGCG 與 hBMSCs 共培養，探討 EGCG 是否可抑制 MG 對 hBMSCs 所誘發之氧化壓力與細胞凋亡，並進一步探討其對hBMSCs 硬骨與脂肪分化能力之影響。結果發現，隨著 MG 濃度提高，hBMSCs 活性受到明顯的抑制；隨著 EGCG 濃度提高，可使細胞活性上升。將 MG、EGCG 與 hBMSCs 共培養後發現，EGCG 能夠提高 hBMSCs 受到 MG 抑制之存活率與三磷酸腺苷 (ATP) 含量；減少受到 MG 誘發的活性氧分子 (ROS)，提高 MG 抑制的過氧化氫酶 (CAT) 與超氧化物歧化酶 (SOD) 活性；抑制受到 MG 所誘發的染色質濃縮現象、並降低 sub-G1 時期與 DNA 片段化增加。提高 MG 抑制硬骨分化之 ALP 與鈣沈澱含量，提高 MG 抑制硬骨分化之鹼性磷酸酶 (ALP)、RUNX2 與第二型骨生成蛋白質 (BMP2) 基因表現，抑制 MG 誘導脂肪分化之三酸甘油酯 (TAG) 含量，抑制 MG 誘導脂肪分化基因之 脂聯素、脂蛋白脂解酶 (LPL) 與 PPARγ 表現。本研究證實 EGCG 具有保護 hBMSCs 的效應，可以減低 MG 的不良影響，並且調節硬骨與脂肪受到 MG 影響的分化能力。
並列摘要
Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis often found at high levels in blood from diabetic patients. MG accumulated in the body forms irreversible advanced glycation end products (AGEs) with DNA and protein, and leads to cell damage. It therefore plays an important role in diabetic complications. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in catechins, can remove many types of free radicals, effectively capture MG molecules and inhibit the formation of AGEs. This study aimed to investigate that MG-induced oxidative stress and apoptosis in human bone marrow mesenchymal stem cells (hBMSCs), and to study whether EGCG can inhibit the effect of MG on hBMSCs. The effects of different concentrations of MG and EGCG on the viability of hBMSCs were first analyzed, respectively. hBMSCs were next cultured in the medium containing both MG and EGCG to study whether EGCG could inhibit the cytotoxic effects and cell apoptotic levels induced by MG. The influence of MG on osteogenic and adipogenic capabilities of hBMSCs was also investigated. The results showed that the viability of hBMSCs decreased as the concentration of MG increased, and cell viability increased as the concentration of EGCG increased. When hBMSCs were incubated in the medium including both MG and EGCG, EGCG was found to increase hBMSCs viability and intracellular adenosine triphosphate (ATP) level, to decrease reactive oxygen species (ROS) level, and to increase catalase (CAT) and superoxide dismutase (SOD) activities. Chromatin condensation, sub-G1 cell population and DNA fragmentation induced by MG addition were downregulated by ECGC. In addition, EGCG enhanced alkaline phosphatase (ALP) activity, calcium deposition as well as gene expressions of ALP, RUNX2 and BMP2 repressed by MG during osteogenesis. Adipogenic markers such triacylglyceride accumulation, and gene expressions of adiponectin, lipoprotein lipase and PPARγ upregulated by MG were suppressed by EGCG. This study demonstrated that EGCG can provide cytoprotective effect in hBMSCs against MG, and further modulate osteogenic and adipogenic differentiation of hBMSCs affected by MG.