Wnt信号通路参与心梗后心肌组织愈合过程的调控

【作者】 陈丽君；

【导师】 左伋； 王克强； 夏蓓莉；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 心肌梗死是严重危害人类健康的心脏病。而心脏破裂则是急性心肌梗死(AMI)致命的并发症,是导致AMI病人死亡的主要原因之一。多数心脏破裂发生在急性心梗的第一周内,且破裂部位多在新鲜梗死区。近年来,器官修复成为一个新的研究方向,心肌梗死区本身的修复过程已引起学者的关注。 急性心梗后,心肌组织的修复是以不完全再生的方式完成的,促进梗死区的愈合以提高心梗患者存活质量,是心梗治疗的新思路。但是,心肌损伤修复过程中诸多结构方面改变的机制目前还知之甚少,除了早期发生的心肌细胞坏死、凋亡和炎症浸润外,可以肯定的是,成纤维细胞以及血管内皮细胞、血管平滑肌细胞的增殖、迁移起到了至关重要的作用。 Wnt蛋白及其受体frizzled介导的信号通路是无脊椎动物和脊椎动物发育过程中起关键作用的信号转导通路之一,它参与了细胞增殖、分化、凋亡和细胞定位控制等过程。在脊椎动物的心脏发育过程中,也发现了Wnt信号转导途径的几个成员的表达。心脏病理过程中胚胎基因的重新表达是一种被普遍观察到的现象。 为了了解Wnt信号通路对急性心肌后梗死区愈合过程的可能调控机制,本研究从整体动物及细胞两个水平深入探讨了该信号通路与心肌梗死愈合的关系。 在建立大鼠急性心肌梗死模型基础上,用免疫组化和原位杂交方法检测大鼠急性心梗后心肌组织Wnt信号转导通路中两个关键的信号分子β-连环蛋白(β-catenin)和Dishevelled-1(Dvl-1)的表达变化,为Wnt通路在心梗愈合过程中的调控作用寻找直接证据。结果表明,心肌梗死后4天β-catenin在梗死区和边缘区的表达开始增加;7天时,其表达水平达高峰;14d后表达减弱,直至消失。β-catenin的上游信号分子Dvl-1的表达先于β-catenin,梗死后1天,Dvl-1 mRNA分子就可以很容易的在梗死边缘区被检测到,尽管此时尚不能检测到蛋白分子的表达。在心梗发生后4天,Dvl-1的表达显著升高并一直处于上调状态直到梗死后2周。在心梗14天后,Dvl-1的表达逐渐减弱,到28天时,已经降低到不能被检测出的水平。本部分研究结果显示在大鼠急性心梗后,β-catenin和Dvl-1的表达变化进程正好符合急性心梗后颗粒组织形成的时间进程,从而提示这些信号分子可能参与调控了心梗后心肌组织愈合过程中肌纤维母细胞以及血管内皮细胞的增殖和迁移等细胞生物学活动,为证实Wnt信号通路参与调控心梗后组织愈合过程提供了在体实验的证据。 为了进一步阐明Wnt信号通路调控成纤维细胞以及血管内皮细胞在该病理生理过程中所表现的细胞生物学行为的分子机制,需要在体外模拟在体Wnt信更多还原

【Abstract】 Myocardial infarction (MI) is one of the leading causes of morbidity and mortality in the whole world. Restoration of the blood supply to the infarcted area and prevention of cardiac remodeling to improve the cardiac function have been the subjects of study for many years. Recently, studies in animals and humans pointed out the importance of the infarct itself as a potential target for intervention, so more attention has been paid to the wound healing process after MI.Although, the mechanisms that govern the architectural control of the processes involved in infarction healing remain to be elucidated, it has been suggested that the migration and proliferation of cells, including myofibroblast and endothelial cells may contribute to these processes. Myofibroblasts not only take responsibility for the deposition of the extracellular matrix in the granulation tissue but also possess contractile properties that prevent the dilatation of the infarction area. Endothelial cells are thought to be involved in neovascularization process of the infarction area.The Wnt signaling pathway is thought to be functionally conserved in vertebrates and invertebrates and plays an important role in cell proliferation, differentiation, organogenesis and oncogenesis. And several members of the Wnt-frizzled signal transduction pathway were found to be expressed during cardiac development in vertebrates. In cardiovascular pathology, re-expression of a fetal gene expression pattern is a generally observed phenomenon.To verify the assumption that Wnt-frizzled pathway may be involved in the repair process after MI, we designed both in vivo and in vitro experiments. First, we established the disease model, the left descending coronary arteries of rats were ligated to induce MI. Immunohistochemistry SABC method and in situ hybridization were performed to detect the expression of two of the most important signaling molecules β -catenin and Dishevelled-1.The results indicated that on day 4 after MI, p-catenin positive cells appeared in granulation tissue and the border zone of the infarct area; the expression of p-catenin increased significantly at 7 days after MI and returned to levels comparable to the sham samples 28 days later. After MI the expression of Dvl-1 gene precede that of P-catenin and 1 day after MI the Dvl-1 mRNA could be easily detected at the border zone, although the protein was still rather hard to detect at that time. 4 days after MI the expression of Dishevelled-1 increased exclusively; 7 days after MI, it seems that the expression reached its peak, the positive staining evenspread into the endothelial and smooth muscle cells of the newly formed and pre-existing blood vessels in the infarction area; after that the Dishevelled-1 expression decreased abruptly and could hardly be detected 28 days after MI.It seems that the time-dependent expression of Dvl-1 and the formation of granulation tissue matched well after MI in rats. These results make a clue that Wnt signaling pathway maybe involved in the proliferation and migration of myofibroblasts and endothelial cells during the process of wound healing after MI in vivo.To clarify the molecular mechanism that Wnt signaling pathway may control the growth and several phonotypes of fibrolasts and endothelial cells related to the repair process after MI in vitro, we need to establish a Wnt-signaling-pathway-constantly-activating cell model. Rat-1, a kind of Rat fibroblasts was chosen as experimental material. Full length Wnt-3a cDNA was inserted into myc tagged pcDNA 3.1 vector to construct the Wnt-3a mammalian expression vector, this vector was stably transfected into Rat-1 cells, then we detected the expression of myc tagged Wnt-3a protein in the culture medium of the Rat-1 AVnt-3a cells by Western blot.Further, we verified that overexpression of Wnt-3a protein make cytoplasmic P-catenin accumulaltion. Thus, we are certain that we established Wnt-signaling-pathway -constantly-activating cell model successfully.Then, we investigated the regulation of Wnt signaling pathway to the growth and several phenotypes of Rat-1 cells and the expression level of several genes related to those phenotypes.The results showed that when Wnt signaling pathway was activated constantly, Rat-1 cells exhibited morphological changes: grew more densely as a monolayer, adopted an elongated and refractile appearance, forming cord-like bundles lined up in a uniform direction. The results of MTT assay and FCM analysis indicated that more Rat-l/Wnt-3a cells enter into G2-phase and the proliferation rate of the Rat-1/ Wnt-3a cells increased significantly compared to the non-transfected cells. Though the migration of Rat-1/Wnt-3a cells increased slightly by the method of Transwell migration assay, there was no statistic significance compared to the non-transfected cells. The result of in vitro scrape wound healing assay showed that for Rat-l/Wnt-3a cells the time course of wound healing decreased significantly.And for the Rat-l/Wnt-3a cells, the expression level of proliferation related gene CyclinDl increased significantly, and the expression level of ECM degradation related gene MMP2 decreased significantly, and the expression level of ECM deposition related gene Fibronectin increased exclusively.For this更多还原