PAR1在斑马鱼淋巴管内皮前体细胞命运决定中的作用和分子机制的研究

The dysfunction of lymphatic vessels is associated with lymphedema, obesity and tumor metastasis et al. It has been found that PROX1 is crucial for the initiation and maintenance of lymphatic fate. Up to now, only a limited numbers of factors have been identified to regulate lymphatic cell fate by regulating PROX1 activity. ..PAR1 (also named as thrombin receptor) plays an important role in the regulating vascular homeostasis. However, the role of PAR1 in lymphatic vessel remains unknown. ..In this application, we here propose that par1 potentially regulates lymphatic fate during zebrafish lymphatic development. In on our preliminary data, we have generated par1 mutant and found that par1 is required for zebrafish lymphatic development. To determine the effect of par1 in zebrafish lymphatic fate, we will generate Tg(prox1a:KalTA4;10xUAS:Venus) line in which lymphatic progenitor cells will be labeled with prox1a:GFP. Meanwhile, to determine whether par1 upstream protease induce par1-mediated lymphatic fate in zebrafish, we will generate mmp13b mutant and Tg(hsp70l:mmp13) line. Here, our preliminary data show that gnai2a, as par1 downstream effector, is also required for zebrafish lymphatic development. To further determine its mechanism, we will check whether Creb directly binds pro1a promoter and regulates its activity. ..Together, in case that this application is successfully funded and that we have finished these experiments, it will offer us a possibility that PAR1 clinic drug can be used for the treatment of lymphatic-related diseases.

在淋巴管发育和命运决定过程中，PROX1是必不可少的。迄今只发现为数不多的因子在PROX1上游调控淋巴管发育。PAR1受体在维持血管动态平衡等方面扮演了重要作用，但其在淋巴管发育过程中的作用从未被报道。. 我们的前期结果表明par1功能缺失能抑制斑马鱼淋巴管发育，且通过细胞实验证实PAR1可以调控PROX1的表达。据此，我们提出假设：par1可能调控斑马鱼淋巴管内皮前体细胞的命运。我们将通过基因敲降、敲除和转基因等技术，结合激光共聚焦成像手段研究par1在斑马鱼淋巴管内皮前体细胞命运决定中的作用。接着，我们将鉴定参与PAR1信号通路，且调控斑马鱼淋巴管发育过程的上游蛋白酶和下游小G蛋白。最后，我们将阐明有关蛋白酶是否诱导斑马鱼淋巴管发育，以及是否通过小G蛋白下游转录因子直接作用于PROX1。. 本项目的顺利实施将为以PAR1为靶点的临床药物治疗淋巴管相关疾病提供理论基础。

淋巴前体细胞的分化是淋巴管生成的关键步骤。然而，其潜在机制仍不清楚。在这里，我们发现非经典的蛋白酶激活受体1（par1）信号通路调控斑马鱼淋巴管前体细胞命运分化。par1功能的丧失通过下调脊索旁淋巴管母细胞中prox1a的表达而损害淋巴管分化，并导致斑马鱼胸导管形成受损。同时，par1下游效应器G蛋白gnai2a是斑马鱼淋巴管发育必需的，其突变模拟了par1突变体中观察到的淋巴管发育缺陷表型。有趣的是，斑马鱼淋巴管发育需要的是mmp13而不是凝血酶。此外，通过遗传相互作用实验证实在斑马鱼淋巴管发育过程中mmp13b是par1上游蛋白酶。在机制上，par1促进后躯干静脉中flt4的表达和磷酸化Erk1/2活性。总之，我们的研究结果表明par1在斑马鱼胚胎淋巴管内皮前体细胞分化过程中扮演了重要的作用。