請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28822
標題: | CYR61 調控骨型蛋白-2 (BMP-2) 致小鼠造骨細胞分化之探討 Cysteine Rich Protein 61 Regulates Bone Morphogenetic Protein-2-dependent Differentiation in Murine Osteoblasts |
作者: | Jean Chiou 邱勤 |
指導教授: | 郭明良(Min-Liang Kuo) |
關鍵字: | 造骨細胞,分化, CYR61,BMP-2,osteoblast,differentiation, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | Cysteine rich protein 61 (CYR61) 為CCN蛋白家族的一員,是一種生長因子,此一生長因子在文獻報導中,有調控細胞發育 (development) 、增生 (proliferation) 、移行 (migration) 及分化 (differentiation)的功能。過去的文獻指出,CYR61在間質幹細胞 (mesenchymal stem cell) 分化成骨骼時會被調控而減少,但是在骨折修復時則會大量表現,顯示其在骨骼發育中之重要性。
骨質代謝包括了骨合成 (bone formation) 與骨消溶 (bone resorption) 。調控骨再塑 (bone remodeling) 的兩種主要細胞為:造骨細胞 (osteoblasts) 及破骨細胞 (osteoclasts) ,造骨細胞主要負責骨骼的製造,破骨細胞則負責骨質的分解及再吸收的作用。當任何一種細胞太多或太少而造成體內不平衡時就可能造成病變,例如骨質疏鬆症 (osteoporosis) 或骨質石化症 (osteopetrosis) 。在骨頭受損斷裂需要癒合時,造骨細胞會移動到傷處進行分化造骨的動作。據報告指出,骨形成蛋白-2 (bone morphogenetic proteins) 可以促進間質幹細胞往硬骨方向分化,促進骨質增生,並且在造骨細胞的移行中扮演重要的角色。另有報告指出,CYR61會透過Wnt signaling pathway 調控bone formation,但BMP-2及CYR61兩者之間的訊息傳遞路徑還有待繼續研究。 因此,本實驗乃以CYR61重組蛋白處理小鼠造骨細胞 (MC3T3-E1),研究CYR61對造骨細胞分化中所扮演的角色。實驗結果發現,將CYR61重組蛋白處理造骨細胞,觀察到隨著CYR61處理濃度增加,可以促進造骨細胞的增生、移動;隨著處理時間的延長可以促使細胞礦物質化(mineralization)。RT-PCR及Western blot分析,可以觀察到bone morphogenetic protein-2 (bmp-2) 的mRNA以及蛋白質表現隨著rCYR61處理時間增加而漸增,利用BMP-2的中和抗體處理細胞再加入CYR61重組蛋白,發現細胞的增生 (proliferation)、移行(migration) 、分化 (differentiation) 的行為有隨著抗體濃度增加而有被抑制的現象。此外在訊息傳遞的探討中,我們發現CYR61重組蛋白可以活化ERK的訊息傳遞路徑,而化學抑制劑 (U0126及PD98059) 則可抑制CYR61誘發之BMP-2表現。此外,integrin alphavbeta3中和抗體可以顯著抑制ERK以及BMP-2的活化表現,甚至可抑制CYR61誘發細胞增生、移行、及分化的行為。綜合以上實驗結果,CYR61可透過integrin alphavbeta3再經由活化ERK訊息傳遞路徑來刺激細胞BMP-2基因表現,並促使造骨細胞分化。 Bone metabolism includes bone formation and reabsoption, which is executed and regulated by osteoblasts and osteoclasts. The dysregulation of these two types of cells would cause bone disorders. When healing, osteoblast migrates to the injury site and proceeds with differentiation process. According to the previous studies, bone morphogenetic protein-2 (BMP-2) may promote mesenchymal stem cells differentiated into bony structure. Cysteine rich protein (CYR61), a member of CCN family, regulates cell development, proliferation, migration, and differentiation. It’s been shown that CYR61 plays an important role in bone development and healing, and CYR61 is regulated by Wnt3A and promotes osteoblasts differentiation. While Wnt signaling pathway is crucial for BMP-2 to induce new bone formation, the regulation mechanism between CYR61 and BMP-2 in bone formation and differentiation is still unclear. Our current data showed that CYR61 significantly increased osteoblasts proliferation and migration in a dose-dependent manner, and increased mineralization in 14 days after treatment. Mechanistically, CYR61 enhanced bmp-2 mRNA and protein expression in time- and dose-dependent manners, and osteoblast migration, proliferation, and differentiation abilities were significantly inhibited by BMP-2 neutralizing antibody. Furthermore, we found that CYR61 induced BMP-2 expression through MAPK/Erk signal pathway. The use of pharmacological inhibitors revealed that ERK was involved in CYR61-mediated BMP-2 expression. Besides, we also showed that integrin alphavbeta3 neutralizing antibody could inhibit CYR61 activated ERK phosphorylation, BMP-2 protein expression, and osteoblast proliferation, migration, and differentiation. Taken together, our results provide evidence that rCYR61 up-regulates BMP-2 mRNA and protein expression, and promoted proliferation, migration, and differentiation through activation of ERK signaling pathway by binding to integrin alphavbeta3 receptor in preosteoblast-like cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28822 |
全文授權: | 有償授權 |
顯示於系所單位: | 毒理學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-96-1.pdf 目前未授權公開取用 | 2.63 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。