2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645739摘要：骨髓中含有可以自我更新及具有多向分化能力的幹細胞-間質幹細胞(MesenchymalStem Cell)，其可分化為造骨細胞、軟骨細胞、脂肪細胞或其他細胞；以及造血幹細胞(Hematopoietic Stem Cells)其可分化為血球細胞、破骨細胞或其他細胞。在一型糖尿病鼠的骨骼中被發現具有成熟造骨細胞減少而脂肪細胞增加的現象。糖尿病狀態下懷孕已知具有引起胎兒先天性畸形的高風險，而此種骨骼系統缺陷包含有生長遲緩及軟骨細胞分化變異。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(bone turnover)。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。依醣類來源不同與蛋白質反應形成之AGEs形式也有所不同，不同形式之AGEs引發之生物效應也可能有所不同。AGEs包含多種化學結構之組成， 但許多研究顯示糖尿病時蛋白質的N-ε-carboxy-methyl-lysine (CML)修飾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。在臨床、糖尿病動物模式及糖尿病腎病變情況的研究上，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(high glucose)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。我們的預試驗結果顯示已可由小鼠骨髓間質及造血幹細胞分化成各骨系細胞及脂肪細胞；且外加AGEs或CML或高血糖狀態時，脂肪細胞分化會受到影響，造骨細胞分化會被抑制；在糖尿病鼠亦可看到AGEs及HbA1c含量增加。預試驗結果提供了本研究可行性及進一步分子機制探討的基礎。計畫分三年進行由細胞模式到實驗動物模式的系列研究：第一年：以細胞實驗模式探討不同形式AGEs (AGE-1, AGE-2, AGE-3)或其主要組成CML處理或高葡萄糖狀態(比較性研究)對於骨髓幹細胞分化成各骨系細胞及脂肪細胞的影響；配合使用RAGE neutralizing antibody或antioxidants，並進行測定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來探討這個課題。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RAGEneutralizing antibody或antioxidants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來探討這個課題。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 neutralizingantibody或antioxidants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: Bone marrow contains stem cells that can differentiate along mesenchymal andhematopoietic pathways during in vitro cultivation, when given adequate conditions.Mesenchymal stem cells (MSCs) are the common progenitors for osteoblasts, chondrocytes,adipocytes, and others; hematopoietic stem cells (HSCs) are the common progenitors forblood cells, osteoclasts, and others. Hyperglycemia may result in many changes in bothgene expression and protein function, which together contribute to the pathogenesis ofdiabetic complications, including diabetic osteoporosis. Diabetic osteoporosis and aging areassociated with a decrease in the number and activity of osteoblast and a parallel increasein the number of adipocyte. On the other hand, the products of nonenzymatic glycation andoxidation of proteins, the advanced glycation end products (AGEs), form under diversecircumstances such as aging, diabetes, and kidney failure. AGEs comprise a large numberof chemical structures, but biochemical and immunohistochemical studies have suggestedthat CML modifications of proteins are 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. Our preliminary data have shown that osteoblastogenesis,chondrogenesis, and adipogenesis from MSCs, and osteoclastogenesis from HSCs, wereobserved in specific culture conditions. We also found that AGEs, CML, andhyperglycemia markedly affected the adipocyte differentiation, whereas it suppressed theosteoblast differentiation. The increased serum AGEs and HbA1c levels could also bedetected in streptozotocin-induced diabetic mice. These results supply the basis to performthis research project. Therefore, the main items to be performed in this research project areto evaluate the pathophysiological effects of AGEs and its major component CML anddiabetic hyperglycemia on the differentiations of bone-related cells from bonemarrow-derived stem cells and its possible molecular mechanisms. This project possessesthree-year duration including: (1) in vitro cellular model: first-year project, to investigatethe actions and molecular mechanisms of AGEs (AGE-1, AGE-2, AGE-3) and its maincomponent CML and high glucose on the differentiation of bone-related cells andadipocytes from bone marrow stem cells in the presence or absence of RAGE neutralizingantibody or antioxidants; the molecular signals including oxidative stress-related signalingsand cell differentiation-related signal molecules were investigated. (2) in vivo animalmodels: second- and third-year, animal models for AGEs/CML treatment andstreptozotocin-induced diabetic hyperglycemia, respectively, to investigate the effects andmolecular mechanisms of AGEs/CML or hyperglycemia on bone-related cells andadipocytes differentiation from stem cells in the presence or absence of RAGE neutralizingantibody or antioxidants or aminoguanidine (AGEs formation inhibitor) or insulin; themolecular signals including oxidative stress-related signalings and celldifferentiation-related signal molecules were investigated. These studies may help us tounderstand the possible pathophysiological effects and molecular mechanisms ofAGEs/CML and diabetic hyperglycemia on the differentiation of bone marrow stem cells,and may provide as an important reference on academic or clinical researches.高糖化終產物骨髓幹細胞分化高糖化終產物受體