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研究生:程文慧
研究生(外文):Wen-Huei Cheng
論文名稱:微管在大鼠心肌細胞間隙接合形成過程中之角色探討
論文名稱(外文):Role of microtubule in gap junction formation of rat cardiomyocytes
指導教授:吳建春吳建春引用關係
指導教授(外文):Jiahn-Chun Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:解剖學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:72
中文關鍵詞:心肌細胞微管間隙接合
外文關鍵詞:microtubuleCx43gap junctionb-tubulinZO-1b-catenin
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摘要

心肌細胞間隙接合的組成蛋白質connexin43(Cx43),會與ZO-1和微管之間有直接的連結,並且與黏附接合的附屬蛋白質b-catenin間接的相接。在先前本實驗室有關大鼠心肌細胞的研究中顯示,當培養在低鈣的環境時這些蛋白質並不存在於細胞接合上,若將細胞轉換至含有正常鈣離子環境中誘導間隙接合的形成,則這些蛋白質會相互連結並形成複合體,由細胞質運送到細胞膜上。而近年來的研究指出在其他種類細胞裡,Cx43必須依賴微管來運輸到細胞膜上。然而有關在心肌細胞中,微管及其他相接蛋白質在Cx43運送到細胞膜之過程所扮演的角色尚未釐清。本研究利用降溫及colchicine來瓦解微管,以taxol來使微管集結成束,或利用brefeldin A(BFA)處理破壞高基氏體後,再利用免疫螢光染色來觀察微管、ZO-1、b-catenin與Cx43在心肌細胞中分佈情形。降溫2小時之後大部分的微管瓦解,而Cx43在細胞接合處的染色,由原本緻密呈線狀的分佈,轉變成較為分散的形式,並伴隨細胞質中出現較多點狀分佈。而回溫之後30分鐘,大部分微管的染色恢復正常,細胞膜Cx43的染色分佈會逐漸恢復。而ZO-1在降溫及回溫的過程中,在細胞接合及細胞質中的變化與Cx43相似,且兩者有同位分佈的情形。然而以colchicine處理6小時,除了與降溫處理發生類似的變化外,去磷酸化的Cx43(Cx43-NP)及ZO-1也會堆積在核周圍。長時間taxol的處理使微管集結成束,會讓Cx43、ZO-1及b-catenin在細胞質點狀染色增加,並干擾間隙接合的分佈。單獨加入BFA破壞高基氏體會使Cx43在細胞膜上的分佈減少。而若在降溫過程中加入BFA,將會阻礙在回溫之後Cx43在細胞接合處的分佈。BFA處理使ZO-1在細胞接合處變得不連續,而在降溫處理中加入BFA也同樣會阻礙在回溫過程中ZO-1運送到細胞膜上,並且ZO-1與Cx43之間有部分同位分佈的情形。而b-catenin在所有的處理中所受到的影響最小,其在細胞接合處的染色分佈都沒有明顯變化;單獨降溫或降溫過程中加入BFA後,其在細胞質中分佈會增加,但回溫之後則恢復正常。在colchicine處理下,b-catenin在細胞質中的點狀染色並不會增加。綜合以上的結果,Cx43運送到膜上需要完整的微管。降溫或藥物處理破壞微管,都會抑制Cx43組裝到細胞膜上,並使部分的Cx43與ZO-1同位分佈於細胞質中。降溫破壞微管之後對Cx43運送到膜上的抑制作用是可逆的,當溫度回復之後,Cx43又可正常的運送到膜上。若以colchicine將微管完全瓦解,則Cx43-NP與ZO-1在核周圍堆積並會抑制間隙接合的形成。以BFA破壞高基氏體會使Cx43在細胞膜上的分佈減少,然而降溫合併BFA處理,即使溫度回復之後,Cx43亦無法正常運送到胞膜上,這些結果顯示完整的微管與高基氏體是心肌細胞形成間隙接合所必需的。
abstract

Cx43 is the major gap junction protein in cardiomyocytes and can bind directly with ZO-1 and microtubule, or indirectly with b-catenin. Previous study of rat cardiomyocytes demonstrated that Ca2+ switch, by shifting 〔Ca2+〕in culture medium from 5 mM to 1.8 mM, induces redistribution of b-catenin, ZO-1, and Cx43 from cytosol to plasma membrane. Recently studies showed that the transportation of Cx43 to cell membrane requires microtubule. However, the role of microtubule and the relationship between microtubule, Cx43, ZO-1, and b-catenin during the assembly of gap junction in cardiomyocytes is unclear. In the present study, cardiomyocytes were treated with ice or colchicine to depolymerize microtubule, taxol to bundle microtubule, or brefeldine A (BFA) to disrupt Golgi apparatus. The distribution of Cx43, ZO-1, b-catenin, and microtubule was assessed by immunofluorescence microscopy. Most of the microtubules were depolymerized 2 h After ice treatment and a diffused junctional and punctate cytosolic staining of Cx43 and ZO-1 was detected in cardiomyocytes. Microtubule reassembled and Cx43 and ZO-1 redistributed to cell junction, when cardiomyocytes were allowed to recover at 37℃ for 15 to 120 min. Treatment of cardiomyocytes with colchicine for 6h induced the accumulation of Cx43-NP and ZO-1 at perinuclear area. Treatment with taxol induced formation of microtubule bundle and also resulted in a punctate staining of Cx43, ZO-1, and b-catenin in cytosol. Treatment with BFA blocked the temperature recovery-induced redistribution of Cx43 and ZO-1 to cell junction after ice treatment. Although ice treatment alone or ice treatment in the presence of BFA increased the punctate staining of b-catenin in cytosol, both treatments had little effect on the staining of b-catenin in cell junction. Treatment with colchicine had little effect on either junctional or cytosolic distribution of b-catenin in cardiomyocytes. In conclusion, these results suggest that disruption of microtubule or Golgi apparatus may result in the disassembly of gap junction in cardiomyocytes and the assembly of Cx43 to cell membrane requires intact microtubule and Golgi apparatus.
目錄

中文摘要……………………………………1
英文摘要……………………………………3
緒論…………………………………………5
材料與方法…………………………………14
結果…………………………………………24
討論…………………………………………31
參考文獻……………………………………42
圖及圖片說明………………………………50
參考文獻

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