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Organ level Inhibition of Inter cellular communication by mutant connexin expression vector

突变连接蛋白表达载体对细胞间通讯的器官水平抑制

基本信息

批准号：
14570198
负责人：
OYAMADA Yumiko
金额：
$ 1.92万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Gap junctions are specialized cell-cell junctions that form intercellular channels and mediate the direct transfer of low molecular weight metabolites and ions. Intercellular communication via gap junction is believed to play important roles in the control of cell growth, differentiation and maintenance of homeostasis. Recently, it has been reported that several human hereditary diseases such as Charcot-Marie-Tooth disease, non-syndromic sensorineural deafness and skin diseases are caused by point mutations of gap junction protein (connexin) genes. However, the pathological processes of these diseases due to abnormalities in gap junctions are poorly understood.In the present study, we have established a method for visualization of cellular function in living cells, while identifying the localization of connexins in real-time. Expression vectors that contain fusion proteins of green fluorescent protein (GEP) and, either wild-type or a dominant-negative mutant connexin43 (Cx43) were cons … More tructed and transfected into primary neonatal rat cardiomyocytes and into communication-deficient HeLa cells. Intercellular communication was estimated by microinjection of gap junction-permeable fluorescent dye (Alexa 568,m.w. 730). Intracellular calcium dynamics in cardiomyocytes were monitored by a fluorescent calcium indicator (Fura Red) in combination with confocal scanning microscopy. Wild-type Cx43-GFP made functional gap junctions in otherwise communication-deficient Hela cells. In contrast, the mutated Cx43-GFP Inhibited dye coupling among primary neonatal rat cardiomyocytes in a dominant-negative manner. The mutated Cx43-GFP induced desynchronization of calcium transients among beating cardiomyocytes with a significantly higher frequency than in wild-type Cx43-GFP. These results indicate that dominant-negative Cx43 can induce inhibition of synchronous beating among cardiomyocytes through desynchronization of calcium transients, and suggest that inhibition of intercellular communication via gap junction might cause arrhythmia and contraction disturbance in the heart. Cx-GFP expression vectors provides a useful systems for studies of localization and function of gap junctions in vivo. Less

缝隙连接是形成细胞间通道并介导低分子量代谢物和离子的直接转移的特化细胞-细胞连接。通过缝隙连接的细胞间通讯被认为在控制细胞生长、分化和维持稳态中起重要作用。最近有报道称，Charcot-Marie-Tooth病、非综合征感音神经性耳聋和皮肤病等多种人类遗传性疾病都是由间隙连接蛋白（连接蛋白）基因的点突变引起的。然而，这些疾病的病理过程，由于异常的间隙连接知之甚少。在本研究中，我们已经建立了一种方法，在活细胞中的细胞功能的可视化，同时确定在实时定位的连接蛋白。将含有绿色荧光蛋白（GEP）和野生型或显性阴性突变型连接蛋白43（Cx43）的融合蛋白的表达载体转染到大肠杆菌中， ...更多信息构建并转染到原代新生大鼠心肌细胞和通讯缺陷的HeLa细胞中。通过显微注射间隙连接可渗透的荧光染料（Alexa 568，m.w. 730）。用荧光钙指示剂Fura Red结合共聚焦扫描显微镜监测心肌细胞内钙动力学。野生型Cx43-GFP在其他通信缺陷的Hela细胞中形成功能性间隙连接。相反，突变的Cx43-GFP以显性负性方式抑制原代新生大鼠心肌细胞之间的染料偶联。突变的Cx43-GFP在搏动的心肌细胞中以比野生型Cx43-GFP显著更高的频率诱导钙瞬变的去磷酸化。这些结果表明，显性负性Cx43可以诱导抑制心肌细胞之间的同步搏动，通过钙瞬变的去激活，并建议通过间隙连接的细胞间通讯的抑制可能会导致心律失常和收缩障碍的心脏。Cx-GFP表达载体为在体内研究缝隙连接的定位和功能提供了一个有用的系统。少