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研究生:宋欣錦
研究生(外文):Sung Hsin-ching
論文名稱:細胞黏附蛋白在大鼠心肌細胞發育過程之免疫組織化學研究
論文名稱(外文):An immunohistochemical study of cell adhesion proteins in the developing rat cardiomyocytes
指導教授:吳建春吳建春引用關係王淑美王淑美引用關係
指導教授(外文):Wu JCWang SM
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:1999
畢業學年度:87
語文別:中文
論文頁數:65
中文關鍵詞:肋狀體心肌細胞心肌發育
外文關鍵詞:costamerecadherincateninvinculintalinlaminincardiomyocyte
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肋狀體(costamere)為橫紋肌細胞膜上特化的構造,是肌原纖維之Z-disc與細胞膜接觸的位置。除了成熟的心肌細胞的細胞膜與細胞外基質間具有以integrin/talin複體為基礎的肋狀體,我們最近的研究證實在培養的心肌細胞中尚有一套以N-cadherin/catenin複體為基礎的肋狀體,它們對於肌原纖維生成及維持肌原纖維結構之穩定扮演重要的角色。然而有關此二套肋狀體在大鼠心肌發育過程中的表現及分佈情形則未探究。本研究首先利用免疫轉漬分析,觀察胚胎第15﹑第19天﹑剛出生﹑出生後第1﹑第2﹑第3﹑第4及第8週大白鼠心肌細胞中N-肋狀體(costamere)為橫紋肌細胞膜上特化的構造,是肌原纖維之Z-disc與細胞膜接觸的位置。除了成熟的心肌細胞的細胞膜與細胞外基質間具有以integrin/talin複體為基礎的肋狀體,我們最近的研究證實在培養的心肌細胞中尚有一套以N-cadherin/catenin複體為基礎的肋狀體,它們對於肌原纖維生成及維持肌原纖維結構之穩定扮演重要的角色。然而有關此二套肋狀體在大鼠心肌發育過程中的表現及分佈情形則未探究。本研究首先利用免疫轉漬分析,觀察胚胎第15﹑第19天﹑剛出生﹑出生後第1﹑第2﹑第3﹑第4及第8週大白鼠心肌細胞中N-cadherin﹑a-catenin ﹑b-catenin ﹑g-catenin﹑vinculin﹑talin及laminin等細胞黏附相關分子的表現。結果顯示N-cadherin﹑a-catenin 及b-catenin在出生後大鼠心肌細胞的表現有逐漸下降的趨勢,vinculin與laminin在出生後的表現逐漸增加,而g-catenin與talin則無明顯趨勢變化。其次以雙重免疫螢光染色觀察心肌細胞發育過程中N-cadherin/catenin及integrin/talin複體的分佈情形,結果顯示在胚胎第15天,此二種複體都散佈在細胞膜上,但分佈的位置不同,少數N-cadherin/catenin複體呈現肋狀體染色。到胚胎第19天, integrin/talin複體開始在細胞側面有較明顯的肋狀體染色。此二種複體分別形成肋狀體的結構,直到出生後第3週都可以觀察到規律整齊排列在細胞外側不同的位置上。從出生後第4週到第8週,細胞膜上僅觀察到由integrin/talin複體構成的肋狀體,至於N-cadherin/catenin為基礎的肋狀體已經不存在,而發育過程中此二種複體分別和vinculin的部份染色位置相同。本研究結果顯示在大鼠心肌發育過程中,N-cadherin/catenin為主的肋狀體出現在早期的發育階段,這可能和心肌發育及肌原纖維之生成有關。而由integrin/talin複體構成的肋狀體在整個發育過程中一直都存在,且在發育愈晚期排列愈整齊,可能扮演著維持心肌細胞結構完整的角色。cadherin﹑a-catenin ﹑b-catenin ﹑g-catenin﹑vinculin﹑talin及laminin等細胞黏附相關分子的表現。結果顯示N-cadherin﹑a-catenin 及b-catenin在出生後大鼠心肌細胞的表現有逐漸下降的趨勢,vinculin與laminin在出生後的表現逐漸增加,而g-catenin與talin則無明顯趨勢變化。其次以雙重免疫螢光染色觀察心肌細胞發育過程中N-cadherin/catenin及integrin/talin複體的分佈情形,結果顯示在胚胎第15天,此二種複體都散佈在細胞膜上,但分佈的位置不同,少數N-cadherin/catenin複體呈現肋狀體染色。到胚胎第19天, integrin/talin複體開始在細胞側面有較明顯的肋狀體染色。此二種複體分別形成肋狀體的結構,直到出生後第3週都可以觀察到規律整齊排列在細胞外側不同的位置上。從出生後第4週到第8週,細胞膜上僅觀察到由integrin/talin複體構成的肋狀體,至於N-cadherin/catenin為基礎的肋狀體已經不存在,而發育過程中此二種複體分別和vinculin的部份染色位置相同。本研究結果顯示在大鼠心肌發育過程中,N-cadherin/catenin為主的肋狀體出現在早期的發育階段,這可能和心肌發育及肌原纖維之生成有關。而由integrin/talin複體構成的肋狀體在整個發育過程中一直都存在,且在發育愈晚期排列愈整齊,可能扮演著維持心肌細胞結構完整的角色。

Costameres, the specialized structures underneath the sarcolemma of straited muscle, are thought to anchor the Z-discs of the peripheral myofibrils to the sarcolemma. Integrin/talin-based costameres have long been found to exist in adult cardiomyocytes. In a previous study, we have demonstrated the existance of the N-cadherin/catenin-based costameres in cultured cardiomyocytes, where they play an important role in maintaining the normal organization of myofibrils and myofibrillogenesis. However, the spatial relationship between integrin/talin-based and N-cadherin/catenin-based costameres during cardiomyocyte development is not known. In this study, immunoblot analyses were used to determine the expression of N-cadherin, a-catenin, b-catenin, g-catenin, vinculin, talin, and laminin in embryonic days 15 (E15), 19 (E19), postnatal day 0 (P0), 1 (1W), 2 (2W), 3 (3W), 4 (4W), and 8 (8W) week old rat hearts. Immunoblot analyses showed that N-cadherin, a-catenin, and b-catenin were down-regulated after birth, vinculin and laminin were up-regulated, and no significant change was found in g-catenin and talin. By using immunofluorescence microscopy, we observed the distribution of N-cadherin/catenin and integrin/talin complexes at the same ages mentioned above. On embryonic day 15, these two complexes were found at different locations along the sarcolemma and only few N-cadherin/catenin complexes appeared in costameric arrangement. On embryonic day 19, the integrin/talin complexes were arranged as costameres. These two costameres distributed at different locations along the sarcolemma up to 3 weeks after birth. From 4 weeks to 8 weeks after birth, only integrin/talin-based costameres, but not N-cadherin/catenin-based costamere, were present in the rat cardiac muscle. Vinculin was shown to be colocalized with these two costameres at all ages examined, indicating that vinculin may also be a component of N-cadherin/catenin-based costamere. These results suggest that N-cadherin/catenin-based costameres were present only at early stage during cardiomyocyte development and may play roles in the early phase of cardiomyocyte development and myofibrillogenesis. Integrin/talin-based costameres appeared in the early development and persisted after birth. This set of costamere may play an important role in maintaining cardiomyocyte structure.

摘要1緒論5材料與方法11抗體11實驗動物12動物組織製備12冷凍切片的製作13免疫螢光染色13心肌組織均質液之製備14蛋白質濃度測定15電泳分離16免疫轉漬16結果18一﹑N-cadherin﹑a-catenin﹑ b-catenin ﹑g-catenin﹑vinculin﹑ talin﹑laminin在大鼠心肌發育過程之表現18二﹑N-cadherin在發育中大鼠心肌內的分佈19三﹑N-cadherin與catenin在大鼠心肌發育過程中的分佈關係19四﹑Vinculin與b-catenin在發育中大鼠心肌內的分佈關係20五﹑Talin與b-catenin在發育中大鼠心肌的分佈關係21六﹑Laminin與talin在大鼠心肌發育過程中的分佈關係22七﹑N-cadherin與laminin在大鼠心肌發育過程中的分佈關係23討論25一﹑兩套肋狀體和vinculin的關係25二﹑N-cadherin/catenin為基礎的肋狀體和肌原纖維的生成有關26三﹑Integrin/talin為基礎的肋狀體和肌肉收縮的關係29四﹑Integrin/talin為基礎的肋狀體和肌原纖維的生成有關30結 論32參 考 文 獻33圖片說明41圖1. 發育過程中大鼠心肌萃取液coomassie brilliant blue的染色分析42圖2. 發育過程中大鼠心肌萃取液的N-cadherin﹑a-catenin﹑ b-catenin ﹑g-catenin﹑vinculin﹑talin及 laminin之免疫轉漬分析44圖3. 大鼠發育過程中N-cadherin在心肌的免疫螢光染色46圖4. 胚胎第15天﹑出生後2週及8週的大鼠心肌中 N-cadherin與a-catenin之雙重免疫螢光染色48圖5. 胚胎第15天﹑出生後2週及8週的大鼠心肌中 N-cadherin與b-catenin之雙重免疫螢光染色50圖6. 胚胎第15天﹑第19天及出生後一週的大鼠心肌中vinculin及b-catenin之雙重免疫螢光染色52圖7 .出生後2週﹑3週﹑4週及8週大鼠心肌中vinculin及b-catenin之雙重免疫螢光染色54圖8. 胚胎第15天﹑第19天及出生後1週的大鼠心肌中talin及b-catenin之雙重免疫螢光染色56圖9. 出生2週大鼠心肌中talin及b-catenin之雙重免疫螢光染色58圖10.出生後3週﹑4週及8週大鼠心肌中talin及b-catenin之雙重免疫螢光染色60圖11.胚胎第15天﹑剛出生﹑出生後2週及8週的大鼠心肌中laminin及talin之雙重免疫螢光染色62圖12.胚胎第15天、剛出生﹑出生後2週及8週的大鼠心肌中N-cadherin及laminin之雙重免疫螢光染色64

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