妊高症主动脉血管内皮与平滑肌细胞中ETBR的功能及调控机制的研究

The vascular dysfunction caused by pregnancy-induced hypertension (PIH) endangers the health of patients. However, the mechanism is not clear, effective preventive measures in clinical also lack.Our research group bases on previous studies which found that in pregnant rats with reduced uterine perfusion pressure (RUPP), the expression of ETBR in aortic vascular endothelial cells was decline obviously,however, in smooth muscle cells,the result was completely opposite,linked with the fact that ETBR in endothelial cells has vasodilator effect, but the role of EBTR in smooth muscle cells was not clear,either the regulation mechanism of ETBR in these two kinds of cells, hypothesed that ET-1 may regulates the expression of ETBR in aortic vascular endothelial cells during pregnancy-induced hypertension by ubiquitination in protein level and by Nrf2 / β-catenin in transcriptional level, ET-1 also cause imbalance of oxidative stress in endothelial cells and lead to the activation of inflammasome, which increases release of IL-1β and IL-18 that induces the expression of ETBR in smooth muscle cells by the activation of NF-κB signaling pathway.This study intends to investigate the regulatory mechanism of ETBR using modern molecular biology techniques in clinical samples, animal models and celluar models, provide a theoretical basis for the mechanism of vascular lesions during and after PIH. This study will provide in-depth understanding of vascular lesions for PIH,own significant theoretical and practical value.

妊高症引起的血管功能障碍长期危害患者的健康，但其机制尚不清楚，目前还缺乏有效的预防措施。课题组基于前期研究发现即妊高症大鼠模型中，ETBR在主动脉血管内皮细胞表达显著下降，而在平滑肌细胞则显著升高，结合国内外研究现状即血管内皮细胞中ETBR具有舒张血管的功效，但其在平滑肌细胞中的作用尚不明确，且两者分别的调控机制也不清楚，提出如下假说：妊高症中ET-1作用于主动脉血管内皮细胞，可能通过泛素化和Nrf2/β-catenin这两种途径分别在蛋白水平和转录水平调控ETBR的表达；并导致内皮细胞氧化应激失衡引起炎症小体激活，通过炎症因子IL-1β和IL-18激活平滑肌细胞的NF-κB信号通路，使平滑肌细胞中ETBR的表达上调。本项目拟使用分子生物学技术，从临床样本、动物模型以及细胞模型三个层面深入研究ETBR的调控机制，为妊高症期间及其后期的血管病变机制提供理论基础，具有重要的理论价值和现实意义。

妊高症引起的血管功能障碍危害患者的健康，但其机制尚不清楚。课题组在前期研究基础上提出“妊高症中ET-1作用于主动脉血管内皮细胞，可能通过泛素化和Nrf2/β-catenin这两种 途径在蛋白水平和转录水平调控ETBR的表达；导致内皮细胞氧化应激失衡引起炎症小体 激活，通过炎症因子IL-1β和IL-18激活平滑肌细胞的NF-κB信号通路，使平滑肌细胞中ETBR 的表达上调” 假说并开展研究。.主要有以下发现：.⑴ 检测妊高症和正常孕妇血浆中ET-1、IL-1β和IL-18的含量，结果提示孕高症组ET-1、IL-1β、Il-18表达均明显升高；检测主动脉中ETBR的mRNA的表达水平，妊高症组（RUPP）明显高于正常妊娠（NP）组；⑵检测尿液中BUN的浓度，RUPP组明显高于NP组；⑶RUPP组大鼠血中IL-18 、ET-1，IL-1β、BUN 含量均高于NP组；⑷采用siRNA敲减Nrf2、β-Catenin，过表达载体过表达Nrf2、β-Catenin，ROS清除剂预处理后，再用ET-1刺激内皮细胞，流式检测ROS，qPCR和WB法检测ETBR的水平，发现ET-1刺激增加ETBR表达，Nrf2过表达后抑制ETBR表达，ROS清除剂抑制ETBR表达。 ⑸RUPP组大鼠主动脉组织ETBR、NLRP3、pro-Caspase1、Caspase-1、pro-IL-1β、IL-1β、pro-IL-1、IL-18 、β-Catenin蛋白表达高于NP组，Nrf2蛋白表达NP组；⑹HE结果显示RUPP大鼠动脉组织厚度和均匀度明显低于NP组、RUPP组大鼠动脉组织胶原含量明显低于NP组；⑺IF双标结果显示RUPP组大鼠主动脉组织ETBR、Rab-7表达高于NP大鼠组；RUPP组大鼠主动脉FAB-11含量明显高于NP组。. 上述发现为妊高症血管病变机制提供理论基础，具有重要的理论价值和现实意义。