血管平滑肌肌浆网不同区域RyR2与RyR1/RyR3间信号“串话”在休克血管双相反应性形成中的作用及机制研究

The vascular reactivity to vasoactivator increased in the early stage while decreased in the late stage after hemorrhagic shock, which was named as biphasic vascular reactivity. Our previous research showed that the activation of RyR2-meidated Ca2+ release from sarcoplasmic reticulum (SR) contributed the developmemt of biphasic vascular reactivity, but the mechanisms involved still remain uncertain. RyR1 and RyR3, distributed at different area of SR in vascular smooth muscle cell,closely associated with the regulation of vascular tone. In current study, we suspected that RyR2 contributed to the development of vascular bi-phasic reactivity through cross-talk with RyR1 and RyR3 located at different area of SR after hemorrhagic shock. Briefly,FKBP12.6-Ca2+/CaM distributed in nuclearlemamal of SR involved in the cross-talk between RyR2 and RyR1,which contributed to the development of vascular hyperreactivity after hemorrhagic shock through coupling with the CaMKII-MLCK dependent signal cascade;FKBP12/Ca2+ located at plasmalemamal of SR associated with the cross-talk between RyR2 and RyR3, which contributed to the development of vasuclar hyporeactivity after hemorrhagic shock through coupling with sorcin/PS2/BKCa dependent signal cascade.The aim of current study is to explore the cross-talk between RyR2 and RyR1/RyR3 distributed in different area of SR in development of vascular biphasic reactivity after hemorrhagic shock and its mechanisms involved through such techniques as laser co-focal, co-IP,tension measurement of VSM and VSMC, patch clamp, etc, and to find out the new molecular target to restore the vascular reactivity after shock.

失血性休克后血管反应性呈双相变化，但机制不清。我们的前期工作显示，RyR2介导的内钙释放促成休克血管双相反应性形成；分布于VSMC肌浆网不同区域的RyR1及RyR3在血管张力调节中发挥不同作用，推测RyR2通过偶联肌浆网不同区域RyR1及RyR3间信号"串话"，促成休克血管双相反应性形成，即：肌浆网近核区FKBP12.6-Ca2+/CaM介导RyR2与RyR1间信号"串话"，促成休克早期血管高反应性形成；肌浆网近质膜区Ca2+/FKBP12介导RyR2与RyR3间信号"串话"，促成休克晚期血管低反应性形成。本研究拟采用RNA干扰、激光共聚焦、IP、血管及细胞张力测定、膜片钳等技术，探讨RyR2与RyR1/RyR3间信号串话在休克血管双相反应性形成中的作用及其机制,旨在阐明肌浆网RyRs介导内钙释放异常与失血性休克后血管反应性双相变化间关系，从新的角度寻求休克血管反应性的调节靶点。

本实验室前期研究发现，失血性休克血管对血管活性物质呈“双相”反应。血管平滑肌细胞（VSMC）肌浆网由RyR2介导钙库内钙参与休克后血管“双相”反应性的形成，但机制不清。本项目采用失血性休克、缺氧诱导血管平滑肌及VSMC“双相”反应性模型，通过基因转染等技术证实：失血性休克大鼠肠系膜上动脉VSNC肌浆网不同亚型RyRs在休克血管“双相”反应性形成中起不同作用，即：由RyR1的过度激活促成休克早期血管高反应性的形成，RyR3的过度激活促成休克晚期血管低反应性的形成，RyR2的过度激活可能参与休克血管双相反应性的形成。.我们还观察了失血性休克早期大鼠肠系膜上动脉平滑肌RyR2/RyR1间的相互串话及其在休克血管高反应性形成中的作用，发现：在失血性休克早期，大鼠肠系膜上动脉血管平滑肌RyR2-FKBP12.6复合子表达显著降低，同时伴随着RyR2-CaM复合子的表达显著降低，RyR1-CaM复合子的表达显著升高，提示CaM可能参与休克早期RyR2与RyR1间的“信号串话”。我们进一步观察失血性休克晚期大鼠肠系膜上动脉血管平滑肌RyR2/RyR3间相互串话及其在休克血管低反应性形成中的作用，发现：在失血性休克晚期，大鼠肠系膜上动脉平滑肌sorcin-RyR2复合子表达逐渐降低，胞浆sorcin浓度显著升高，FKBP12-RyR3复合子形成显著升高；sorcin在VSMC胞浆的表达可能与休克血管对NE诱导收缩反应性呈显著负相关；sorcin siRNA转染明显恢复缺氧VSMC对NE诱导的收缩反应性；此外，在失血性休克晚期的大鼠肠系膜上动脉，PS2-BKCa β亚基复合子表达明显升高，但PS2不与BKCa α亚基形成复合子；在正常的血管平滑肌细胞，转染sorcin-EGFP质粒可显著上调PS2-BKCa β亚基复合子的表达，且具有钙依赖性；在缺氧3 h的血管平滑肌细胞，转染sorcin siRNA可显著抑制由缺氧诱导的PS2-BKCa β亚基复合子表达升高。结果显示，sorcin是参与RyR2/RyR3间信号串话及血管低反应行形成的重要信号分子。.本项目基本明确了在失血性休克不同时相，VSMC亚型RyRs间的“信号串话与休克血管双相反应性形成间的关系。通过对对本项目的研究，已发表论著4篇，其中SCI（IF：2.798）论著1 篇。