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Analysis of coronary vascular development in an animal model of congenital heart disease - in vivo and in vitro studies to detect a role of proepicardial organ derived cells

先天性心脏病动物模型中冠状血管发育的分析 - 体内和体外研究以检测心外膜器官衍生细胞的作用

基本信息

批准号：
16390299
负责人：
NAKAGAWA Masao
金额：
$ 7.1万
依托单位：
Shiga University of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

We examined distributional patterns of quail-derived proepicardial organ (PEO) cells in the hearts of chick-quail chimeric embryos produced following the procedure described by Manner. Quail PEO cells formed epicardium at day 5 after incubation, beneath which epithelial-mesenchymal transformation (EMT) of PEO cells were observed. Quail PEO cells also were detected in the myocardium and around the future ostia of coronary arteries at day 6 and at day 9 after incubation, respectively. In the mouse embryos at the coincident developmental stage with chick, tenascin-C was expressed in PEO but was suppressed when PEO cells moved to the cardiac surface, migrating into the myocardium. Tenascin-C expression was upregulated in EMT. Mouse PEO cells cultured on the collagen gels in the presence of fetal calf serum formed tube structures. RT-PCR analyses of RNAs extracted from cultured PEO demonstrated expression of various vascular growth factors including bFGF, HGF, VEGF, Flt-1, Flk- 1, Ang-1, Ang-2 and Tie-2, however, the expression of these growth factors were downregulated when bis-diamine was added into the culture medium. In rat embryos, vascular plexus was formed between the epicardium and myocardium at 14.5 embryonic day (ED) and vascular structure was recognized in the myocardium at ED 15.5. The vascular plexus was dispersed and vascular structures were not formed in the embryonic hearts when bis-diamine was given to mother rats at ED 10.5. Immunohistological studies showed delayed expression of the vascular growth factors in those rat embryos treated with bis-diamine.These results suggested that bis-diamine caused the abnormal development of coronary vasculature by making a direct effect on growth of PEO cells and by disturbing EMT and expression of vascular growth factors.

我们研究了鹌鹑衍生的前心外膜器官（PEO）细胞在鸡鹌鹑嵌合体胚胎的心脏中的分布模式。鹌鹑PEO细胞在孵育后第5天形成心外膜，在心外膜下观察到PEO细胞的上皮间质转化（EMT）。在孵育后第6天和第9天，分别在心肌和冠状动脉未来开口周围检测到鹌鹑PEO细胞。在与鸡发育阶段一致的小鼠胚胎中，腱生蛋白-C在PEO中表达，但当PEO细胞移动到心脏表面，迁移到心肌中时被抑制。Tenascin-C在EMT中表达上调。在胎牛血清存在下，在胶原凝胶上培养的小鼠PEO细胞形成管状结构。RT-PCR分析显示，培养的PEO中有bFGF、HGF、VEGF、Flt-1、Flk- 1、Ang-1、Ang-2和Tie-2等多种血管生长因子的表达，但加入双二胺后，这些生长因子的表达均下调。在大鼠胚胎中，心外膜和心肌之间的血管丛形成于胚胎14.5天（艾德），心肌中的血管结构在胚胎艾德15.5天被识别。在艾德10.5时给予母鼠双二胺，胚胎心脏血管丛分散，血管结构未形成。免疫组化结果显示，bis-diamine处理的大鼠胚胎中血管生长因子的表达延迟，提示bis-diamine通过直接影响PEO细胞的生长，干扰EMT和血管生长因子的表达，导致冠状动脉血管发育异常。