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研究生:林文智
研究生(外文):Wen-Zhi Lin
論文名稱:探討破骨細胞經由抑鈣素刺激之後所引起的足體重新配置及封閉環脫離的作用中PYK2/Src所扮演的角色為何
論文名稱(外文):The Role of PYK2/Src in Calcitonin-induced Podosome Reassembly and Sealing Zone Detachment in Osteoclasts
指導教授:徐佳福史 中
指導教授(外文):Jia-Fwu ShyuChung Shih
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:2005
畢業學年度:93
語文別:中文
論文頁數:85
中文關鍵詞:破骨細胞足體抑鈣素
外文關鍵詞:osteoclastspodosomesealing zoneαvβ3 integrincalcitoninPyk2/Src/Cbl
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破骨細胞是從血緣性的幹細胞最終分化而成的多核細胞,其在骨吸收的過程裡扮演著一個不可或缺的角色。為了要進行骨吸收作用,破骨細胞經由一個特別的黏著構造稱之為足體(podosome)來黏附在細胞外基質或骨表面形成了一個富涵F-actin的環狀構造,那這個環狀構造相當於正在進行骨吸收之破骨細胞的sealing zone。許多的實驗證據顯示,PYK2/Src/Cbl這個複合體對於integrin對足體所調控的相關訊息傳遞,具有決定性的影響。經由研究PYK2/Src/Cbl這個複合體,已經可以深入地說明integrin所調控的訊息傳遞有關於outside-in及inside-out的這個觀念。
抑鈣素是由32個胺基酸所構成的氨基化合物,它會抑制破骨細胞的活動力,促使細胞收縮,並且會將由actin所構成的環狀構造給瓦解。因此可以合理的假設在足體裡的PYK2/Src/Cbl這個複合體可能是抑鈣素所引起的相關訊息傳遞的主要作用目標。先前我們實驗室已將從兔子分離出來的破骨細胞培養於含有細胞外基質的細胞培養皿上,然後用抑鈣素刺激之後發現PYK2會去磷酸化,並且會有重新分佈的情形產生。PYK2去磷酸化預期會阻止PYK2/Src/Cbl這個複合物的形成,因此而影響到破骨細胞的活動力及黏著能力。然而有關於抑鈣素對於破骨細胞PYK2/Src/Cbl這個複合體的功能及分佈的影響並不是很清楚。我們因此使用了免疫沉澱法及西方點墨法來研究是否抑鈣素會對Src與PYK2之間的結合以及Src的磷酸化造成影響。結果顯示,經由抑鈣素刺激之後會使得破骨細胞Src的磷酸化增加,也會使得Src跟PYK2之間的結合增加。此外,我們也經由使用免疫螢光化學染色法分析發現,抑鈣素會使得在sealing zone裡的PYK2去磷酸化,並且也會使得細胞中央的Src與PYK2的結合增加。更進一步,我們使用Src酪氨酸416及酪氨酸527磷酸化的抗體來做實驗,結果發現抑鈣素會增加酪氨酸416而不是酪氨酸527的磷酸化。此外經由共軛焦顯微鏡的觀察,我們也發現經由抑鈣素刺激之後,Src酪氨酸416的磷酸化及Src酪氨酸527的去磷酸化,主要發生在破骨細胞的中央部位。最後我們得到一個結論說,抑鈣素可能是藉著減少破骨細胞sealing zone裡PYK2的磷酸化及增加細胞中央Src酪氨酸416的磷酸化和Src與PYK2之間的結合,而促使細胞的足體重新配置以及sealing zone脫離骨表面。
Osteoclasts (OCs) are multinucleated, terminally differentiated cells which play an essential role in bone resorption. To resorb bone, OCs attach to extracellular matrix or the bone surface via specialized attachment structures called podosomes, which form a prominent F-actin-rich ring that is thought to correspond to the sealing zone of resorbing OCs. It had been showed that Pyk2/Src/Cbl complex is critical to the αvβ3 integrin-mediated signaling in podosome. The concept of outside-in and inside-out of integrin-mediated signaling has been elucidated in depth through the study of Pyk2/Src/Cbl complex.
Calcitonin (CT) is a 32-amino acid polypeptide which inhibits OC motility, induces OC retraction, and disrupts the actin-ring structure of OCs. Thus it is reasonable to assume that the Pyk2/Src/Cbl complex in podosome could be the potential target for the CT-induced signaling. In isolated authentic rabbit OCs cultured on extracellular matrix-coated Petri dish, we previously showed that CT induced dephosphorylation and redistribution of Pyk2. The dephosphorylation of Pyk2 would be expected to prevent the formation of Pyk2/Src/Cbl complex and therefore inhibit OC motility and attachment. However, the effects of CT on Pyk2/Src/Cbl complex function and their distribution in OCs are not clear. We therefore investigated whether CT affects Pyk2/Src association and Src phosphorylation in OCs by using the immunoprecipitation and Western blot methods. The result showed that CT induced an increase of Src tyrosine phosphorylation and Pyk2/Src association. Using immunofluorescent confocal analysis, we showed that CT induces dephosphorylation of Pyk2 in the sealing zone and increase Src tyrosine phosphorylation and Pyk2/Src association in the central region of OCs. Further examination using specific Y416 and Y527 phosphorylation antibodies of Src showed that CT induced increase of Y416 but not Y527 phosphorylation. The increase of Y416 and decrease of Y527 phosphorylation were found in the central region of OCs after CT stimulation. In conclusions, CT may induce podosome reassembly and sealing zone detachment by decrease Pyk2 phosphorylation in the sealing zone and increase Y416 phosphorylation of Src and Pyk2/Src association in the central region of OCs.
正文目錄...I
圖目錄...V
中文摘要...Ⅵ
英文摘要...Ⅷ
第一章 緒言...1
第一節 研究背景...1
第二節 破骨細胞...4
壹、破骨細胞型態及功能...4
貳、足體...5
參、Integrin...7
一、Integrin結構...7
二、Integrin的活化及訊息傳導機轉...7
三、破骨細胞的Integrin...9
第三節 抑鈣素...11
壹、抑鈣素的發現...11
貳、抑鈣素的種類及化學結構...11
參、抑鈣素的生理及藥理作用...12
一、抑鈣素對骨組織的作用...12
二、抑鈣素對腎臟的作用...13
三、抑鈣素對中樞神經系統的作用...13
四、抑鈣素臨床療效 ...14
肆、抑鈣素受體...15
伍、抑鈣素在細胞株內訊息傳遞...15
一、腎臟近曲小管LLC-PK1細胞株...15
二、腎臟HEK-293細胞株...16
陸、抑鈣素在破骨細胞內訊息傳遞...17
第四節 PYK2...19
壹、細胞內訊息傳遞簡介...19
貳、酪胺酸蛋白激酶次家族:PYK2和FAK...19
參、PYK2在細胞內訊息傳遞...21
肆、PYK2和pl30Cas在破骨細胞扮演的角色...22
第五節 Src...23
壹、Src Family Proto-oncogenes...23
貳、Src的結構...24
參、Src的調節...24
肆、Src的功能...25
伍、Src對破骨細胞的作用...26
第六節 研究的動機與目的...28
第二章 材料與方法...30
第一節 實驗材料...30
第二節 實驗方法...33
壹、破骨細胞的取得 ...33
貳、蛋白質標準曲線的建立及破骨細胞蛋白質濃度定量...34
參、免疫沈澱法...34
肆、硫酸十二酯鈉聚丙烯銨膠體電泳法...34
伍、西方點漬法...35
陸、免疫螢光化學染色法...37
柒、破骨細胞脫離分析...38
第三章 結果...39
第一節 破骨細胞在含有第一型膠原蛋白培養皿上黏貼的情形...39
第二節 抑鈣素促使破骨細胞產生收縮、脫離的情形...39
第三節 抑鈣素對於破骨細胞PYK2與P-PYK2分佈情形的影響...39
第四節 抑鈣素對於破骨細胞Src酪胺酸磷酸化的影響...40
第五節 抑鈣素對於破骨細胞PYK2與Src association的影響...40
第六節 抑鈣素對於破骨細胞Src與PYK2 association的影響 ...41
第七節 抑鈣素對於破骨細胞Src與P-PYK2分佈情形的影響...41
第八節 抑鈣素對於破骨細胞Phospho-Src (Tyr 416)磷酸化的影響...42
第九節 抑鈣素對於破骨細胞Src與Phospho-Src (Tyr 416)分佈情形的影響...42
第十節 抑鈣素對於破骨細胞Phospho-Src (Tyr 527)磷酸化的影響...43
第十一節 抑鈣素對於破骨細胞Src與Phospho-Src (Tyr 527)分佈情形的影響...43
第四章 討論...56
第一節 抑鈣素造成破骨細胞的收縮及脫離...56
第二節 抑鈣素降低破骨細胞PYK2的磷酸化而導致細胞脫離的現象...56
第三節 抑鈣素增加破骨細胞內中心區域Src酪氨酸磷酸化的現象...57
第四節 抑鈣素增加破骨細胞內中心區域Src與PYK2結合的現象...58
第五節 抑鈣素增加破骨細胞內中心區域Phospho-Src (Tyr 416)的量以及促使細胞中心還有細胞外圍少部份Phospho-Src (Tyr 527)瓦解的情形...58
第五章 結論...61
參考文獻...62











圖目錄
頁次
圖1.破骨細胞在含有第一型膠原蛋白培養皿上黏貼的情形...... 45
圖2.抑鈣素促使破骨細胞產生收縮、脫離的情形 46
圖3.抑鈣素對於破骨細胞PYK2與P-PYK2分佈情形的影響 47
圖4.抑鈣素對於破骨細胞Src酪胺酸磷酸化的影響 48
圖5.抑鈣素對於破骨細胞PYK2與Src互相結合的影響 49
圖6.抑鈣素對於破骨細胞Src與PYK2互相結合的影響 50
圖7.抑鈣素對於破骨細胞Src與P-PYK2分佈情形的影響 51
圖8.抑鈣素對於破骨細胞Phospho-Src (Tyr 416) 磷酸化的影響
52
圖9.抑鈣素對於破骨細胞Src與Phospho-Src (Tyr 416)分佈情形的影響 53
圖10.抑鈣素對於破骨細胞Phospho-Src (Tyr 527)磷酸化的影響
54
圖11.抑鈣素對於破骨細胞Src與Phospho-Src (Tyr 527)分佈情形的影響 55
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