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標題: | 油酸對於大鼠心肌細胞之黏附接合的影響 Effects of Oleic Acid on Adherens Junction in Rat Cardiomyocytes |
作者: | Han-Row Fan 范涵柔 |
指導教授: | 王淑美 |
關鍵字: | 油酸,心肌,黏附接合, Oleic acid,Cardiomyocytes,Adherens junction, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 先前的研究結果顯示,油酸會抑制大鼠心肌細胞間隙接合的組合和細胞間的溝通。由於心間盤的黏附接合可以調節間隙接合的組合,這引發我們去探討油酸的處理是否也影響心肌細胞中cadherin-catenin複合物的穩定性。我們的實驗結果顯示,隨著油酸(100 μg / ml)處理的時間越久,N-cadherin、α-catenin、β-catenin及p120-catenin在細胞接合處的染色分布及強度越少。在黏附接合上這些蛋白質的減少伴隨著細胞質內N-cadherin、β-catenin及p120-catenin點狀染色增加,我們也以early endosomal marker去確認這些N-cadherin的點狀染色是否為內吞小囊,但證明其中部分為早期內吞小囊。另外,油酸的處理會引起細胞內Protein kinase Cα(PKCα)、PKCε及Src的短暫活化。抑制PKCα及PKCε的活性可以抑制油酸所引起的Src活化,故得知Src位於PKCα及PKCε的下游。細胞若以Gö6976 ( PKCα抑制劑 )、εV1-2 ( PKCε抑制劑 ) 或PP2 ( Src 抑制劑 ) 預先處理,則發現三者皆可抑制油酸所引起的黏附接合去組合。從生化分析也證實,N-cadherin、β-catenin及p120-catenin在細胞膜表現量因油酸處理而減少,Gö6976及εV1-2預先處理可阻止油酸所引起的變化。此結果顯示了這三種激酶都參與油酸所啟動的訊息傳遞。我們也以免疫沉澱法證實油酸處理主要活化Src家族中的Fyn。另外,油酸處理也使Fyn(或phospho-Src)和β-catenin及p120catenin結合增加β-catenin及p120-catenin的酪氨酸磷酸化增加,這些變化分別使β-catenin與α-catenin及p120-catenin與N-cadherin的結合減弱。細胞以PP2預先處理抑制了油酸處理引起的β-catenin和p120-catenin的酪氨酸磷酸化,此結果證明油酸所活化的Fyn會造成β-catenin及p120-catenin之酪氨酸磷酸化。除此之外,油酸可能透過活化Casein Kinase 1( CK1 )引起N-cadherin和β-catenin的絲胺酸磷酸化,這種修飾可能加速N-cadherin和β-catenin的分解。總而言之,油酸活化了心肌細胞中多種訊息路徑,造成cadherin及catenin的磷酸化,最後造成黏附接合的去組合。 Our previous studies have shown that oleic acid (OA) inhibits gap junction-mediated intercellular communication and gap junction assembly in rat cardiomyocytes. It has been known that adherens junction (AJ) of intercalated disc can modulate the assembly and disassembly of gap junctions. The aims of this study were to investigate whether OA also modulated cadherin-catenin complex of AJ in neonatal rat cardiomyocytes. By immunofluorescence examination, the staining distribution and intensities of N-cadherin, β-catenin, and p120-catenin at cell-cell contact sites were time-dependently decreased, which was concomitant with the increase in the cytosolic punctate staining in response to OA treatment. Some of these structures were identified to be early endosomes. Immunoblot analyses further demonstrated that OA induced transient activations of PKCα, PKCε, and Src, and the Src kinase appeared to be the downstream target for PKCα and PKCε, respectively, since blockade of PKCα or PKCε activity inhibited the OA-induced activation of Src kinase. Moreover, pretreatment of Gö6976 (PKCα+βⅡ inhibitor), εV1-2 (PKCε inhibitor), or PP2 (Src inhibitor) prevented the OA-induced disassembly of adherens junctions. OA induced the decreased expression of N-cadherin, β-catenin and p120-catenin in the membrane fraction, and this event was prevented by pretreatment with Gö6976 or εV1-2. Thus, our data showed that theses kinase were involved in OA-induced AJ disassembly. We also proved that OA activated Fyn, a member of Src family kinase, by immunoprecipitation. Moreover, OA increased the binding of Fyn to β-catenin and of Fyn to p120-catenin. This led to increase tyrosine phosphorylation of β-catenin and p120-catenin, which subsequently induced the dissociation of β-catenin and α-catenin and of p120-catenin and N-cadherin. Pretreatment with PP2 could prevent Fyn-induced tyrosine phosphorylation of βv-catenin and p120-catenin. This result suggests that Fyn is responsible for the tyrosine phosphorylation of β-catenin and p120-catenin by OA. Futhermore, OA might cause serine phosphorylation of N-cadherin and β-catenin mediated by casein kinaseⅠ(CKⅠ), and this modification may accerlerate the degration of N-cadherin and β-catenin. In conclusion, we identified multiple signaling cascades responsible for the OA induced-AJ disassembly by modification of cadherin and catenin. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37468 |
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顯示於系所單位: | 解剖學暨細胞生物學科所 |
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