2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645943摘要：多種生理和病理情況，以及氧化還原狀態的改變，會影響細胞內質網的蛋白質折疊能力，導致未折疊或錯誤折疊的蛋白質在內質網內部積累，或引發所謂的內質網壓力(endoplasmic reticulum stress)。在內質網壓力持續期間內，CCAAT/enhancer binding protein (C/EBP)同源蛋白 C/EBP homologous protein (CHOP）是屬於最高度正調節性基因之一。數項研究曾表示 CHOP 參與內質網壓力誘導的細胞凋亡過程(apoptosis)。CHOP 基因剔除小鼠曾被發現會表現出骨生成減少情形，但一項有關過度表現 CHOP 的基因轉殖小鼠的研究卻顯示在骨骼微環境中有 CHOP過度表現的情形時，反而會損害造骨細胞而導致骨量缺少症(osteopenia)。我們的預試驗有關動物微斷層掃描(CT)結果也顯示 CHOP基因剔除小鼠的骨密度有明顯減少情形。故 CHOP在骨生成上的調節角色仍存有爭議的，且機制也不清楚。實際上，CHOP 在骨髓幹細胞分化(造骨細胞、破骨細胞、脂肪細胞和軟骨細胞)和骨細胞的生長/功能的調節角色仍然不清楚，有必要進一步加以釐清。因此，本研究計畫是計劃利用CHOP基因剔除小鼠模式經生物體外(ex vivo)或活體實驗方式並配合體外細胞實驗，來探討內質網壓力相關因子-CHOP 在正常和疾病的情況下對於骨代謝的調節角色。計畫分三年完成此項研究計畫：第一年：計劃研究 CHOP 於正常生理狀態下在骨代謝上的調節角色；研究培養的來自野生型或CHOP 基因剔除小鼠的骨髓幹細胞、骨細胞和骨組織在細胞生長、分化和功能上的可能影響和變化；另外也分析這些小鼠在骨/軟骨形態和病理、骨質和骨骼微架構上之變化，以及可能參與的訊息分子；部分實驗也會以培養的正常人類骨細胞或骨髓幹細胞來探討 CHOP 在骨細胞生長及分化上的角色。第二年：計劃探討 CHOP 於糖尿病高血糖狀態下在骨代謝上的調節角色(糖尿病相關骨質疏鬆症)；分析野生型或 CHOP 基因剔除小鼠於糖尿病狀態時對於骨/軟骨形態和病理、骨量和骨骼微架構的影響，以及可能參與的訊息分子。第三年：計劃研究CHOP 於雌激素缺乏誘發骨質疏鬆症之狀態下在骨代謝上的調節角色；分析野生型或 CHOP 基因剔除小鼠於雌激素缺乏時對於骨/軟骨形態和病理、骨質和骨微架構的影響，以及可能參與的訊息分子。本計畫之研究結果預期將可以讓我們了解 CHOP 在正常生理和疾病狀態(糖尿病和雌激素缺乏相關骨質疏鬆症)時，在骨髓幹細胞分化和骨/軟骨代謝上所扮演的可能重要角色，也可提供做為臨床上尋找骨質異常相關疾病治療策略之參考或線索。 <br> Abstract: Various physiological and pathological circumstances as well as alteration in the redox status, can compromise the endoplasmic reticulum (ER) capacity in protein folding, resulting in the accumulation of unfolded or misfolded proteins in the ER lumen, or ER stress. During prolonged ER stress, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is one of the most highly up-regulated genes. Several studies have implicated CHOP in ER stress-induced apoptosis. CHOP null mice have been found to exhibit decreased bone formation; however, a study of transgenic mice overexpressing CHOP in the bone microenvironment showed that CHOP overexpression impairs the osteoblastic function leading to osteopenia. Our preliminary study also showed that bone mineral density was markedly decreased in CHOP-knockout mice determined by μCT. The regulatory role of CHOP in bone formation is controversial. In fact, the regulatory role of CHOP in the cell differentiation (osteoblasts, osteoclasts, adipocytes, and chondrocytes) from bone marrow stem cells and bone cell growth/function still remains unclear and needs further to be clarified. Therefore, this proposal is planned to explore the role of ER stress-related factor CHOP in bone metabolism under normal and disease conditions by the ex vitro and in vivo experiments using a CHOP knockout mouse model and the in vitro studies using bone cell culture model. The following experimental procedures are designed to achieve the specific aims of this proposal. First Year: Plan to investigate the role of CHOP in bone metabolism under normal physiological condition. The cell growth, differentiation, and function in cultured bone marrow stem cells, bone cells and bone tissues isolated from wild-type and CHOP knockout mice will be tested. The morphology and pathology, bone mass, and microarchitecture in these mice, and involved signaling molecules will also be analyzed. In some experiments, the role of CHOP in growth and differentiation of cultured human bone cells or human bone marrow stem cells will be tested. Second Year: Plan to investigate the role of CHOP in bone metabolism during diabetic hyperglycemia (diabetes-related osteoporosis). The bone cell differentiation, bone morphology and pathology, bone mass and microarchitecture, and involved signaling molecules in bone/cartilage of wild-type mice and CHOP knockout mice will be analyzed. Third Year: Plan to investigate the role of CHOP in bone metabolism during estrogen deficiency-induced osteoporosis. The bone cell differentiation, morphology and pathology, bone mass and microarchitecture, and involved signaling molecules in bone/cartilage of wild-type mice and CHOP knockout mice will be analyzed. We anticipate that the results of the proposed studies would show that CHOP may play an important role in bone marrow stem cell differentiation and bone/cartilage metabolism, and would show that the role of CHOP in bone cell differentiation and bone/cartilage metabolism under the disease conditions (diabetes- and estrogen-related osteoporosis). These studies may provide more information for us to understand the related clinical orthopaedic problems and may provide a clue to the solution.C/EBP 同源蛋白（CHOP）內質網壓力骨代謝骨質疏鬆症C/EBP homologous protein (CHOP)ER stressbone metabolismosteoporosis