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Functional analysis of gap junctional intercellular communication using differentiation of embryonic stem cells in vitro

利用胚胎干细胞体外分化进行间隙连接细胞间通讯的功能分析

基本信息

批准号：
08670259
负责人：
OYAMADA Masahito
金额：
$ 1.41万
依托单位：
Kyoto Prefectural University of Medicine (1997)Sapporo Medical University (1996)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1.Connexin expression during cardiomyocytic differentiation of ES cells in vitroThe transcripts for C_x43 and C_x45 were detected in undifferentiated ES cells and in embryoid bodies before and after the appearance of beating cardiomyocytes. In contrast, C_x40 transcripts were not observed in undifferentiated ES cells, and were barely detectable in 3-and 5-day-old embryoid bodies. C_x40 transcripts significantly increased with the appearance of beating cells similar to those of cardiac-specific genes. Dye coupling was present among undifferentiated ES cells, pre-beating cells of embryoid body outgrowth and ES cell-derived beating cardiomyocytes. When dye was injected into a beating cell, dye spread was restricted to neighboring beating cells. Immunofluorescence demonstrated that C_x43 protein was localized not only in beating cells but also in surrounding non-beating cells, whereas myosin heavy chain alpha/beta was exclusively positive in the beating cells. These data suggest that the expression of multiple connexins is differentially regulated during the cardiomyocytic differentiation of ES cells in vitro and that C_x40 expression may be linked to early stages in cardiomyocytic differentiation.2.Cardiomyocytic differentiation of C_x43-deficient ES cells in vitroC_x43-deficient ES cells showed little dye coupling measured by Lucifer Yellow dye transfer assay, but there was cardiomyocytic differentiation in vitro, which was not significantly different from that of wild-type ES cells. RT-PCR revealed that the amounts of C_x40 and heart-specific gene transcripts significantly increased with the appearance of beating cells as with wild-type ES cells, whereas C_x43 transcripts were not detected in the C_x43-deficient cells. These data indicate that C_x43 is not essential for cardiomyocytic differentiation of ES cells in vitro and that expression of other connexins such as C_x40 and C_x45 is not influenced by the absence of C_x43 expression.

在未分化的ES细胞和拟胚体中检测到C_x43和C_x45的转录本。在未分化的ES细胞中未检测到Cx40转录本，在3日龄和5日龄的拟胚体中几乎检测不到Cx40转录子。随着跳动细胞的出现，Cx40转录本显著增加，与心脏特异基因相似。未分化的ES细胞、拟胚体突起的预搏动细胞和ES细胞来源的搏动心肌细胞之间存在染料偶联。当染料被注入到正在跳动的细胞中时，染料扩散仅限于邻近的正在跳动的细胞。免疫荧光显示，CX43蛋白不仅定位于搏动细胞，而且定位于周围的非搏动细胞，而肌球蛋白重链α/β仅在搏动细胞中呈阳性。这些结果表明，在体外ES细胞的心肌分化过程中，多种连接蛋白的表达受到差异调控，Cx40的表达可能与心肌细胞分化的早期阶段有关。2.荧光黄转移实验显示，在vitroc_x43基因缺陷的ES细胞中，Cx43基因缺陷的ES细胞的心肌分化几乎没有染料偶联，但在体外有心肌细胞的分化，这与野生型ES细胞没有显著差异。RT-PCR结果显示，与野生型ES细胞一样，随着搏动细胞的出现，Cx40和心脏特异基因转录本的数量显著增加，而Cx43缺陷细胞中未检测到Cx43转录本。这些结果表明，C_x43在体外对ES细胞的心肌细胞分化不是必需的，其他连接蛋白如C_x40和C_x45的表达不受C_x43表达的影响。