VE-Cadherin 磷酸化介导P120ctn、Kaiso和MMP信号通路参与颅内动脉瘤形成机制研究

The formation mechanism of intracranial aneurysm is still unclear. It was confirmed by our previous study that vascular endothelial injury initiated by VE-Cadherin phosphorylation play an important role in the formation of intracranial aneurysms. The association of VE-Cadherin phosphorylation with P120 catenin shows that VE-Cadherin phosphorylation can regulate P120 catenin function. It has been reported that p120ctn can bind directly with a transcription factor, Kaiso, implicating a possible function as a co-factor for this transcription factor. Worthy of investigation, we found Matrix Metalloproteinase-2(MMP-2), a member of the MMPs family which are involved in proteolytic modeling of the extracelluar matrix, possess two conserved copies of the core sequence-specific Kaiso site. Probably, MMP-2 is the target gene of Kaiso.MMP-2 has been showed to play a critical role in the formation of intracranial aneurysms, however the regulation of MMP-2 gene in endothelial cell is not very clear yet. In this study,we are about to explore the relationship between transcription of MMP-2 and the P120 catenin -Kaiso complex and try to test the hypothesis that P120 catenin, Kaiso,and MMP-2 signal initiated by VE-Cadherin phosphorylation is involved in the aneurysmal formation.

颅内动脉瘤形成机制仍不清楚，本项目在申请人前期发现VE-cadherin磷酸化在其中发挥重要作用和对VE-cadherin磷酸化调控相关因子进行系统分析的基础上，结合p120ctn不仅调控VE-cadherin磷酸化，且能与转录抑制因子kaiso结合参与内皮细胞核内靶基因调控的科研实践，分析与颅内动脉瘤形成相关基因的启动子区域，发现MMP-2启动子系列里含有两个Kaiso能识别的系列，提出VE-Cadherin 磷酸化介导的P120ctn、Kaiso和MMP-2信号通路与颅内动脉瘤形成机制相关的假说。拟应用抑制剂、基因干扰和萤火虫酶检测等技术，通过体内和体外实验，探讨VE-cadherin磷酸化诱导p120ctn，kaiso和MMP-2信号通路在动脉瘤形成中的作用及调控机制，为探明颅内动脉瘤形成机制奠定实验基础和理论依据。

本课题探讨VE-cadherin磷酸化诱导p120ctn，kaiso和MMP-2信号通路在动脉瘤形成中的作用及调控机制。拟应用抑制剂、基因干扰和萤火虫酶检测等技术，通过实验性大鼠颅内动脉瘤模型、家兔颅内动脉瘤模型和体外冲击流场系统（T型流室系统）对颅内动脉瘤形成的血流动力学机制实现体内、体外的模拟，我们发现：（1）动脉瘤的形成过程中，VE-cadherin与p120ctn在血管内膜的表达进行性下降； p120ctn在颅内动脉瘤形成过程中出现类似于β-catenin向细胞核移位，提示p120ctn可能通过与 VE -cadherin分离并向核内转移的机制参与颅动脉瘤形成；（2）冲击流场作用下血管内皮细胞间黏附连接的损害多发生于高WSS 及高WSSG 的区域。血流动力学的改变可以引起血管内皮细胞在形态、生长方式及相关蛋白表达的改变，进而导致血管内皮细胞间黏附连接稳定性的下降及相关致炎因子的表达改变。VE-cadherin磷酸化明显降低了HUVEC细胞间黏附的稳定性；（3）冲击流作用下内皮细胞装置能成功模拟血管分叉处的血流环境。随着在流体动力学作用下内皮细胞培养装置培养时间的延长，HUVEC胞质内p120ctn的表达减少；而细胞核内p120ctn的表达增加，表明在血流动力学作用下，p120ctn可能发生质核穿梭，由胞质进入胞核内；细胞内的Kaiso在胞核表达明显增多。干扰p120ctn基因表达后，Kaiso在胞核表达增多的趋势减弱。上述结论证实了我们提出的“VE-Cadherin 磷酸化可介导P120ctn、Kaiso和MMP信号通路并参与颅内动脉瘤形成”，为探明颅内动脉瘤形成机制奠定实验基础和理论依据。