血管紧张素II-1型受体自身抗体致子痫前期胎盘血管功能失稳态的分子机制研究

Placental vascular dishomeostasis is the core of pathological changes of preeclampsia; however, the underlying mechanisms are unclear. From our previous original discovery, we found that the expressions of large-conductance calcium-activated potassium channels (BKCa) and voltage-gated potassium channels (KV), which can cause vasodilation, were decreased in placental vascular smooth muscle isolated from angiotensin II type 1 receptor (AT1R) autoantibodies (AT1-AA)-positive preeclamptic patients. This study intends to establish BKCa/KV knockout rat model, use patch clamp, single cell tension tester, etc. to clarify whether AT1-AA can inhibit the channels of BKCa/KV in placental vessel through sustained activation of AT1R-PLC-PKC pathway, which will result in increased preeclamptic placental vascular tension; then using radioactive ligand binding assay, fluorescence energy transfer technology, etc. to investigate the molecular mechanism of AT1-AA-induced non-desensitization of AT1R pathway in placental vascular smooth muscle from multiple aspects-decreased phosphorylation of AT1R, changes of vesicular formation proteins or transport proteins, abnormal receptor conformation and the rate of synthesis, reduced cell membrane fluidity. According to the general objectives--to inveatigate the dynamic regulating mechanism .of vascular homeostasis and remodeling this project aimed to reveal the molecular regulatory mechanisms and key node of preeclampsia vascular lesion from the perspective of receptor non-desensitization mediated channel disturbances.

胎盘血管功能失稳态是子痫前期的核心病变，但机理不清。本课题组前期原创性发现，血管紧张素Ⅱ-1型受体（AT1R）自身抗体（AT1-AA）阳性的子痫前期患者胎盘血管平滑肌上介导舒张的大电导钙激活钾通道（BKCa）和电压依赖钾通道（KV）表达下降。本项目拟通过构建BKCa/KV通道敲除大鼠，利用膜片钳、单细胞张力检测等技术，明确AT1-AA通过持久激活AT1R-PLC-PKC信号通路抑制胎盘血管平滑肌BKCa/KV，进而引起血管张力升高的作用；采用放射配体结合实验、荧光共振能量转移等技术，从AT1R磷酸化障碍、内吞囊泡形成及转运蛋白改变、受体构象和合成速率异常、细胞膜流动性下降等角度， 寻找AT1-AA致胎盘血管平滑肌AT1R及下游信号持久激活的关键分子机制。本课题围绕“探讨血管稳态与重构的调控机制”的总目标，从受体不脱敏介导通道紊乱的角度揭示参与子痫前期血管功能失稳态的分子调控机理和关键节点。

血管功能失稳态是子痫前期的核心病变，但机理不清。本课题组前期原创性发现，血管紧张素Ⅱ-1型受体（AT1R）自身抗体（AT1-AA）阳性的子痫前期患者血管平滑肌上介导舒张的大电导钙激活钾通道（BKCa）表达下降，但意义及机制不清。本项目通过BKCa通道敲除大鼠及AT1R敲除大鼠，利用膜片钳、血管环离体培养等实验，明确AT1-AA通过持续激活AT1R导致血管平滑肌细胞BKCa抑制，引起小动脉损伤。利用全内反射荧光显微镜、质膜分离、荧光显微镜成像技术等技术证实AT1-AA通过限制AT1R内吞，致血管持续收缩和胞内钙超载；利用荧光示踪、荧光共振能量转移、全内反射荧光显微镜等技术证实AT1-AA使β-arrestin募集障碍，引起AT1R内吞障碍。利用Real-time PCR、桑格测序、原位杂交、双荧光素酶报告基因分析等实验，明确AT1-AA通过circErbB4-miR-29a-5p-AT2R使AT2R上调造成大动脉重塑。利用等离子共振、Dot-Blot、ELISA、血管环、钙离子成像实验，证实AT1-AA存在多样性，四种不同病理效应AT1-AA与AT1R结合位点不同，需用不同短肽阻断。本研究揭示了自身免疫紊乱机制在血管稳态失衡相关疾病中的重要作用，BKCa通道可能是恢复小动脉稳态的潜在干预靶点。此外，AT1-AA通过限制β-arrestin介导的受体内吞，导致AT1R信号持续活化，提示AT1-AA引起的AT1R信号偏移可能是现有ARB/ACEI类药物存在残余风险的重要原因；不同效应的AT1-AA，可识别AT1R细胞外第二环的不同表位肽段，提示对于临床患者体内的AT1-AA应该进行分类筛查和个体化治疗。

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