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研究生:詹雯惠
研究生(外文):CHAN,Wen-hui
論文名稱:Stomatin過量表現會經由幫助破骨細胞的融合導致骨量流失
論文名稱(外文):Overexpression of Stomatin Induces Bone Loss in Transgenic Mice by Helping Osteoclast Cell Fusion
指導教授:徐佳福
指導教授(外文):Shyu,Jia-Fwu
口試委員:徐佳福王懷詩蕭嘉陽
口試委員(外文):Shyu,Jia-FwuWang,Hwai-ShiShiau,Chia-Yang
口試日期:2012-05-24
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:68
中文關鍵詞:口形蛋白細胞融合破骨細胞
外文關鍵詞:stomatincell fusionosteoclast
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細胞融合在破骨細胞的功能以及生理特性上扮演著重要的角色。當破骨細胞融合後,可以具有吸收骨基質的能力,來維持骨量的恆定。 Stomatin屬於箝入性膜蛋白 (integral membrane protein)且亦分佈於脂筏 (lipid raft) ,分子量約為 31kDa。主要表現在身體的周邊血、肝臟、胎盤、肌肉、骨髓等部位。先前研究指出,當轉染 stomatin至卵巢細胞,使之大量表現 stomatin時,會有較多的多核細胞形成。因此猜測 stomatin在細胞融合成多核的過程中扮演了重要的角色。在動物實驗,藉由 micro-CT以及組織型態測量學分析六個月大的 stomatin過度表達之基因轉殖鼠,其結果顯示骨吸收 (破骨細胞數量上升)以及骨生成皆上升,處於高轉運型骨 (high turn-over bone),但是吸收速率大於生成速率,因此使得 STOM-overexpression的小鼠最後的骨量仍是下降的。為了瞭解 stomatin的功能,將攜帶綠色螢光的stomatin基因轉染至RAW 264.7細胞中。經由 TRAP+以及抑鈣素誘導細胞內 cAMP上升可知,當 stomatin高表達時生成多核破骨細胞的數量上升,並且發現 DC-STAMP以及 CD9表現也上升。以 RANKL刺激 RAW 264.7細胞分化並利用免疫螢光染色分析,發現 stomatin會往細胞膜 lipid raft處移動,並且與actin的表現分佈在相似區域。另一方面,發現 stomatin與 DC-STAMP、 CD9在膜上的表現也分佈在相似區域。總結來說, stomatin過度表達可能會增加與 DC-STAMP、CD9會在細胞膜脂筏處彼此連結,並藉由 stomatin與 actin的連結來幫助調節細胞骨架的移動,進而調節破骨細胞的融合。因為破骨細胞數量的增加,使得骨的吸收作用上升,進而導致骨量下降。
Cell-cell fusion is a crucial step to function and physiology of osteoclasts (OCs). After the fusion step, OCs resorb bone matrix more efficently and maintain the bone remodeling. Stomatin is a 31.5 kDa integral membrane protein or lipid rafts associated protein. The distribution of human stomatin expression is major in peripheral blood, bone marrow, placenta, fetal lung, and fetal liver. Our previous study indicated that stomatin over-expressed CHO (chinese hamster ovary -K1) cells may induce the formation of multinucleated cells through the cell-cell fusion process. This suggests that stomatin may play a role in the process of the multinucleated cell formation. In animal study, the high turn-over bone of STOM-overexpression from 6-month-old B6 mice which bone resorption (osteoclast number) and bone formation rate was increased as compared to control by micro-CT and histomorphometric analysis. The decreased bone density is contributed to higher bone resorption rate than bone formation rate. To study the function of stomatin, we constructed stomatin gene with EGFP gene and transfected it into RAW 264.7 cells. Overexpression of stomatin induced osteoclast formation as demonstrated by positive TRAP stain and increase of calcitonin-induced cAMP, and increased the expression of DC-STAMP and CD9. Using double immunofluorescence staining of RANKL-stimulated RAW cells, stomatin expression was localized at lipid raft membrane microdomain, with high levels of colocalization with the peripheral actin-rich belt. Stomatin were also found colocalization with DC-STAMP and CD9. In conclusion, stomatin overexpression induced osteoclast fusion via increasing association of DC-STAMP and CD9 with lipid raft, and associated with actin helping cell fusion by regulated cell skeleton. The decrease of bone density may due to increase of osteoclastic bone resorption.
正文目錄
正文目錄 ………………………………………………………………I
圖目錄…………………………………………………………………III
附錄目錄 ………………………………………………………………V
中文摘要………………………………………………………………VI
英文摘要 …………………………………………………………VIII
第一章 緒言 …………………………………………………………1
第一節 破骨細胞 (Osteoclast, OC) ……………………………………………1
第二節 破骨細胞生成及分化 (osteoclastogenesis)………………3
第三節 細胞融合 …………………………………………………6
第四節 脂筏 (Lipid raft) ………………………………………………………………14
第五節 Stomatin ………………………………………………………………………………………16
第六節 研究目的與動機 …………………………………………20
第二章 材料與方法 …………………………………………………21
第一節 實驗材料 ……………………………………………………21
第二節 實驗方法 ……………………………………………………27
第三節 動物實驗 ……………………………………………………32
第三章 結果 …………………………………………………………38
第四章 討論 …………………………………………………………43
第五章 結論 …………………………………………………………46
參考文獻 ……………………………………………………………47

圖目
圖1:利用 micro-CT分析控制組與 STOM-overexpression小鼠之骨股以及第五節腰椎………………………………………54
圖2:將控制組與 STOM-overexpression小鼠的股骨以及第五腰椎,利用 micro-CT進行各項骨參數的分析………………55
圖3:利用組織切片來觀察小鼠骨小樑數量的變化……………………………………………………………………………………56
圖4:利用組織型態學方法分析小鼠股骨之非脫鈣骨組織切片………………………………………………………………………57
圖5:利用組織型態學方法分析小鼠股骨之非脫鈣骨組織切片………………………………………………………………………58
圖6:利用骨螢光標示劑 (calcein)的沉積,來分析小鼠骨生成速率之變化 ……………………………………………………59
圖7:RAW264.7細胞株以 50 ng/ml RANKL刺激後,細胞型態的變化以及 stomatin的表現情形……………………………60
圖8:以免疫螢光染色檢測 stomatin在兔子破骨細胞表現情形……………………………………………………………………61
圖9:以免疫螢光染色檢測 stomatin與 actin在 RAW264.7細胞以及在兔子的破骨細胞中表現情形 ………………………62
圖10:利用軟體分析 stomatin與 actin在未分化前以及分化成破骨細胞後,其螢光的 intensity的變化 ………………63
圖11:以免疫螢光染色檢測 stomatin與 flotillin-1在 RAW264.7細胞以及在兔子的破骨細胞中表現情形 ……………64
圖12:利用軟體分析 stomatin與 flotillin-1在未分化前以及分化成破骨細胞後,其螢光的 intensity的變化 ……65
圖13:以免疫螢光染色檢測 stomatin與CD9在兔子破骨細胞及 RAW264.7細胞中表現情形 …………………………………66
圖14:以免疫螢光染色及 western blot檢測 stomatin與 DC-STAMP在兔子破骨細胞及RAW264.7細胞中表現情形 ……67
圖15:以免疫螢光染色檢測 stomatin與 actin在兔子破骨細胞表現情形 ……………………………………………………68

附錄目錄
附圖一:轉染 RAW264.7細胞的TRAP染色細胞數量變化 …………………………………………53
附圖二:RAW264.7細胞經由 stomatin轉殖前後用 calcitonin刺激後 cAMP的變 …………53
附圖三:以免疫螢光染色檢定 RANKL刺激轉染 stomatin與 CD9表現情形 ……………………53
附圖四:以免疫螢光染色檢定 RANKL刺激轉染 stomatin與 DC-STAMP表現情形 ……………53
附圖五:推測的機制圖 ………………………………………………………………………………53

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