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研究生:簡瑞林
研究生(外文):Jui-Lin Chien
論文名稱:探討分析YC-1對於治療骨質疏鬆症的潛力及機制
論文名稱(外文):Analysis Of Therapeutic Potential And Mechanism Of YC-1 For Osteoporosis
指導教授:徐佳福
指導教授(外文):Jia-Fwu Shyu
口試委員:顏茂雄王懷詩
口試委員(外文):Mao-Hsiung YenHwai-Shi Wang
口試日期:2012-05-29
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:69
中文關鍵詞:骨質疏鬆症
外文關鍵詞:YC-1osteoporosis
相關次數:
  • 被引用被引用:1
  • 點閱點閱:244
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  • 下載下載:20
  • 收藏至我的研究室書目清單書目收藏:0
骨質疏鬆症的成人年齡超過55歲是一個重要的公共衛生問題,每年耗資數十億美元。骨質疏鬆症的治療主要是基於使用藥物抑制破骨細胞的骨吸收。尋找最適當的治療藥物及方法是一個緊迫的問題。最近,我們發現YC-1有效抑制PC-12 (老鼠嗜鉻細胞瘤) 細胞的鈣蛋白酶。 YC-1是一類新化合物並在人類血小板和血管平滑肌的研究中確定可以直接激活可溶性鳥苷酸環化酶,並且可以使NO pathway下游的酶對於NO的刺激更為敏感。鈣蛋白酶被認為是促進細胞擴散和運動調節黏著斑 (focal adhesion) 複合物的組裝和拆卸,而在一般有活性的破骨細胞鈣蛋白酶的活性是被需要的。本研究的目的是分析YC-1對於骨質疏鬆症的治療潛力及機制。在成熟的破骨細胞從紐西蘭大白兔的骨髓中分離出來,在加入YC-1 (10nM) 治療後使用rodamine phalloidin 染破骨細胞的機動蛋白環 (actin ting) 並使用共聚焦顯微鏡觀測。在RANKL刺激RAW264.7細胞後再加入YC-1(100 nM)誘導的細胞凋亡,以Western blot分析顯示YC-1促使細胞表現caspase 3和8 細胞凋亡蛋白。破骨細胞使用牙本質骨片上的培養, YC-1 (100nM) 減少兔子破骨細胞對於牙本質骨片的吸收。在3月齡去卵巢引起的骨質疏鬆大鼠皮下注射YC-1(1mg/kg)持續4週由micro CT照影結果顯示YC-1可減少骨量之流失。此外,histomorphomatric的分析,顯示YC-1有提升骨生成的效果。使用西方墨點法分析鈣蛋白酶活性結果表示,YC-1導致破骨細胞的外在凋亡途徑,而抑制破骨細胞的骨吸收或許是藉由Src。總之,YC-1可能有治療骨質疏鬆症的可能性,通過抑制破骨細胞的骨吸收。在未來,我們將進行探討在YC-1對於成骨細胞的治療的效果。
Osteoporosis is a major public health problem for adults over age 55 years costing billions of dollars. The treatment of osteoporosis is largely based on the
use of agents that inhibit bone resorption by osteoclasts. Search for the most appropriate treatment is an urgent issues. Recently, we found that YC-1 can
effectively inhibit calpain in PC-12 cells. YC-1 is a novel class of compounds identified to directly activate soluble guanylyl cyclase, and sensitize the enzyme for NO production in human platelets and in smooth muscle. Calpains are thought topromote cell spreading and locomotion by modulating the assembly and disassembly of the focal adhesion complexes, and calpain activity is required for normal osteoclast activity. The aim of this study is analysis of therapeutic potential and mechanism of YC-1for osteoporosis. In authentic osteoclasts isolated from rabbit bone marrow, 10 nM of YC-1 induced disruption of actin ring as demonstrated by rodamine phalloidin labeling under confocal microscope examination. In Rankl-stimulated raw264.7 cells 100 nM of YC-1induced apoptosis as showed by Western blot analysis of caspase 3 and 8. Using rabbit osteoclasts cultured on the dentin disc, 100 nM YC-1 induced decrease of pit formation. In three-month-old ovariectomy-induced osteoprotic rats, subcutaneously injection of YC-1 (1mg/kg) for 4 weeks caused reduced bone loss as showed by micro CT. Furthermore, histomorphomatric analysis was performed to evaluate the bone remodeling process. Western blot analysis of calpain activity shows that YC-1 cause osteoclasts to the extrinsic apoptotic pathways, and inhibits calpain activity perhaps by Src. In conclusion, YC-1 may have the potential for the treatment of osteoporosis, by inhibiting osteoclastic bone resorption. In the future, we will perform to explore the effect of the YC-1 treat in osteoblast.

正文目錄…………………………………………………………………I
圖目錄…………………………………………………………………………III
中文摘要………………………………………………………………………IV
英文摘要………………………………………………………………………VI
第一章 緒言………………………………………………………………1
第一節 破骨細胞 (The osteoclast) 之簡介…………………………………1
第二節 YC-1 (3-(5'-Hydroxymethy-2'-furyl)-1-benzyl indazole) 之簡介......3
第三節 鈣蛋白酶 (calpain) 簡介 ……………………………………………6
第四節 抑鈣素 (Calcitonin) 簡介……………………………………………7
第五節 燒結焦磷酸二鈣 (sintered dicalcium pyrophosphate, SDCP)
簡介………………………………………………………………11
第六節 細胞凋亡 (Apoptosis) 之簡介……………………………………14
第七節、研究動機與目的………………………………………………… 16
第二章 材料與方法…………………………………………………………17
第一節 實驗材料與藥品……………………………………………17
第二節 培養基與其他溶液之配製……………………………………20
第三節 實驗方法 …………………………………………………………24
第四節 動物實驗………………………………………………………………29
第三章 結果 …………………………………………………………………33
第四章 結果圖片………………………………………………………………40
第五章 討論…………………………………………………………………51
第六章 結論…………………………………………………………………55
參考文獻………………………………………………………………………56
圖目錄
頁次
圖1、 以免疫螢光染色 (Immunofluorescence staining) 來觀察分析破骨細胞在YC-1及其他藥物處理下其存活率和細胞活性……………40
圖2、 以反射干擾顯微鏡 (Interference reflection microscopy) 以及pit formation 來觀察破骨細胞的活性……………………………………42
圖3、骨缺損大鼠以YC-1、鈣素或是 SDCP治療後,其第六腰椎之3 D重組及數據分析…………………………………………………………43
圖4、骨缺損大鼠以YC-1、抑鈣素或是SDCP治療後,其第六腰椎非脫鈣包埋以Villanueva osteochrome bone stain及Toluidine Blue O觀察其型態並加以分析………………………………………………………45
圖5、破骨細胞以YC-1、抑鈣素及SDCP刺激後,活化態 caspase-3、8、9之表現量差異…………………………………………………………47
圖6、破骨細胞以YC-1、抑鈣素與SDCP刺激下,在不同的時間點觀察對鈣蛋白酶活性影響……………………………………………………49
圖7、破骨細胞以YC-1、抑鈣素及SDCP刺激後, 非活化態Src Tyrp527之表現量差異……………………………………………………………50


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