2013-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/672473摘要：糖尿病高血糖狀態曾被指出會降低造骨細胞功能，且促進破骨細胞相關之骨吸收作用；但也有學者指出高葡萄糖狀態會經由降低破骨細胞分化而改變骨更新(boneturnover)。因此，糖尿病高血糖狀態對於骨髓幹細胞分化成各骨系細胞(造骨細胞、軟骨細胞、破骨細胞)及脂肪細胞的詳細作用和機轉仍有待進一步探討及釐清。另一方面，高糖化終產物(advanced glycation end-products, AGEs)是體內醣類與蛋白質、脂質或核酸等大分子在無酵素條件下發生反應後的產物。AGEs 包含多種化學結構之組成， 但許多研究顯示糖尿病時N-ε-carboxy-methyl-lysine (CML)是蓄積在體內最主要的AGEs。AGEs 可通過改變被修飾蛋白的結構和功能或與特異的AGEs 受體(Receptors of AGEs，RAGE)結合影響胞內訊息傳遞、刺激細胞因子等釋放而發揮致病效應。AGEs 與RAGE 的相互反應增加被認為是導致糖尿病慢性併發症的可能原因，包括骨骼系統病變。曾發現AGEs 的累積會降低骨骼強度及增加骨折的危險性。但是AGEs 影響間質幹細胞分化成造骨細胞、軟骨細胞和脂肪細胞以及造血幹細胞分化成破骨細胞的詳細情形及分子機制，以及在糖尿病骨質減少症(osteopenia)上的真正角色仍尚不清楚。本研究計畫之目的在於探討AGEs 及其主要組成CML 對於骨髓幹細胞分化成骨系細胞(造骨細胞、破骨細胞和軟骨細胞)及脂肪細胞之詳細作用及可能之分子機制，並與高葡萄糖或高血糖情況比較，以釐清糖尿病狀態時對於骨髓幹細胞分化影響的作用機制，並嘗試可能之治療策略。正在執行中第一年計劃是以細胞實驗模式探討AGEs 或其主要組成CML 處理對於骨髓幹細胞分化成各骨系細胞及脂肪細胞的影響及分子機制；部分研究成果(2011/08/01-2011/12/15 期間數據)顯示CML 會抑制造骨細胞分化以及脂肪細胞分化，皆可能是透過與RAGE 之結合作用，但是影響之訊息分子卻不相同，在脂肪細胞分化狀態下，主要是透過抑制ERK 磷酸化以及PPAR-γ 活化，此可能與促進ROS 生成有關；而在造骨細胞分化狀態下，則可能是透過iNOS 活化產生NO 進而去抑制p38 磷酸化的表現。第一年部分研究成果提供了本研究可行性及進一步動物實驗模式探討的基礎；因此，我們計畫繼續完成後面兩年之動物實驗部分，以完整達成本研究計畫之目的及目標。第一年(原第二年)：以正常實驗動物給予適當劑量及處理時間之AGEs 或CML，或合併RAGE 中和抗體或抗氧化劑處理；研究骨質變化及骨髓幹細胞分化成各骨系細胞及脂肪細胞情形，進行測定AGEs 或CML 含量、氧化壓力相關或調控骨系細胞或脂肪細胞分化相關之訊息分子。第二年(原第三年)研究計畫：以streptozotocin 糖尿病鼠實驗模式，或合併insulin 或aminoguanidine (AGE 形成抑制劑)或RAGE 中和抗體或抗氧化劑處理；研究骨質變化及骨髓幹細胞分化成各骨系細胞及脂肪細胞情形，進行測定AGEs 或CML 含量、氧化壓力相關或調控骨系細胞或脂肪細胞分化相關之分子訊息來探討這個課題。希望經由對AGEs 或CML 或高血糖狀態下骨髓幹細胞分化機制的研究及了解，可以提供學術上或臨床研究上之重要參考。<br> Abstract: Diabetic osteoporosis and aging are associated with a decrease in the number and activityof osteoblast and a parallel increase in the number of adipocyte. The products ofnonenzymatic glycation and oxidation of proteins, the advanced glycation end products(AGEs), form under diverse circumstances such as aging, diabetes, and kidney failure.AGEs comprise a large number of chemical structures, but CML modifications ofproteins have been suggested to be predominant AGEs that accumulate in vivo. Anincrease in the interaction between advanced glycation end-products (AGEs) and theirreceptor RAGE is believed to contribute to the pathogenesis of chronic complications ofdiabetes mellitus, which can include bone alterations such as osteopenia. It has beenreported that AGE contributed to diminished bone healing in type 1 diabetes, possiblymediated by RAGE. However, the precise actins and molecular mechanisms of diabetichyperglycemia on the alterations of osteoblastogenesis, chondrogenesis,osteoclastogenesis, and adipogenesis are still unclear. Therefore, the main items to beperformed in this research project are to evaluate the pathophysiological effects of AGEsand its major component CML on the differentiations of bone-related cells from bonemarrow-derived stem cells and its possible mechanisms. First-year project, which isbeing implemented, investigates the actions and molecular mechanisms of AGEs and itsmain component CML on the differentiation of bone-related cells and adipocytes frombone marrow stem cells. The part of results of the first year research project (datacollection during 2011/08/01-2011/12/15) showed that CML is capable of inhibiting theosteoblast and adipocyte differentiations, which are likely through a RAGE-relatedpathway. CML inhibits adipocyte differentiation may be through the ROS-relatedinhibition of ERK phosphorylation and activation of PPAR-γ. CML inhibits osteoblastdifferentiation may be through a NO/iNOS-induced inhibition of p38 phosphorylation.These results supply the basis to perform the animal experiments in second and thirdyears. In vivo animal models: first (original second)- and second (original third)-year,animal models for AGEs/CML treatment and streptozotocin-induced diabetichyperglycemia, respectively, to investigate the effects and molecular mechanisms ofAGEs/CML or hyperglycemia on bone-related cells and adipocytes differentiation fromstem cells in the presence or absence of RAGE neutralizing antibody or antioxidants oraminoguanidine (AGEs formation inhibitor) or insulin; the molecular signals includingoxidative stress-related signalings and cell differentiation-related signal molecules wereinvestigated. These studies may help us to understand the possible pathophysiologicaleffects and molecular mechanisms of AGEs/CML on the differentiation of bone marrowstem cells, and may provide as an important reference on academic or clinical researches.糖尿病高血糖高糖化終產物骨髓幹細胞分化HyperglycemiaAdvanced glycation end-productsStem cell differentiation