Notch3信号通路在肺高压肺血管重构中的作用及机制

Notch signaling is at the convergence point of five pathways: hypoxic HIF1-α signaling, BMP signaling, PDGF signaling, TGF-β1 signaling, and Nuclear factor-κB signaling, all of which have implicated in the development of pulmonary hypertension (PH). Our previous studies indicate that irreversibility of PH associated with congenital heart disease is related to hyperproliferative and anti-apoptotic pulmonary arterial smooth muscle cell (PASMC). We hypothesized that Notch3 signaling involves in the development of pulmonary vascular remodeling in PH. We target PASMC. We will detect expression of Notch3 on lung tissue and analyze its relationship between mean pulmonary arterial pressure and pulmonary vascular resistance. Adeno-Notch3 ICD vector will be constructed and infect PASMC cultured in vitro, then growth rate and expression of Hes5 of PASMC will be evaluated. Next, we will utilize Hes5 siRNA to reduce Hes5 expression and evaluate its effects on proliferation of PASMC. Finally, We will test whether blockade of Notch3 cleavage effectively reverse PH in rats. We aim to investigate effects and mechanisms of Notch3 signaling in PH and provide a novel strategy for treatment of PH.

Notch信号通路处于肺高压5条信号通路交点。我们前期研究结果显示先天性心脏病继发肺高压不可逆与肺动脉平滑肌细胞过度增殖、凋亡不足有关。据此我们提出Notch3信号通路参与肺高压肺血管重构发病机制的假说。本研究以肺动脉平滑肌细胞为靶点，重点研究Notch3信号通路对肺动脉平滑肌细胞的调控及机制。该研究从组织学水平检测Notch3在肺组织表达情况及其与平动脉压、肺血管阻力的关系；然后构建NotchICD载体转染体外培养肺动脉平滑肌细胞，检测肺动脉平滑肌细胞增殖情况及Hes5表达；再用SiRNA 干扰技术阻断Hes5表达，检测肺动脉平滑肌细胞增殖情况及Hes5表达；用Notch抑制剂，通过动物模型验证阻断Notch受体信号通路能否逆转肺血管重构。分析分子间相互影响及相应信号蛋白之间上下游关系。本研究旨在探讨Notch3信号通路在肺高压中的作用和调节机制，为探寻治疗新靶点提供依据。

先心病是导致新生儿死亡和残疾重要原因。先心病肺高压不可逆仍是目前治疗重点和难点。先心病肺高压不可逆与肺动脉平滑肌细胞过度增殖、凋亡不足，导致肺血管重构有关。Notch信号通路处于肺动脉高压5条信号通路交汇点。Notch 信号通路是调控肺动脉平滑肌细胞分化至关重要通路，调控肺动脉平滑肌细胞身份识别、增殖和抗凋亡能力。本研究以肺动脉平滑肌细胞为靶点，重点研究Notch3信号通路对肺动脉平滑肌细胞的调控及机制。从组织学水平检测Notch3在肺组织表达情况及其与平动脉压、肺血管阻力的关系；构建NotchICD载体转染体外培养人肺动脉平滑肌细胞，检测肺动脉平滑肌细胞增殖情况及Hes5表达；用SiRNA 干扰技术阻断Hes5表达，检测人肺动脉平滑肌细胞增殖及凋亡情况及Hes5表达。重要结果：Notch3在肺动脉高压患者及野百合碱和左向右分流肺动脉高压大鼠肺组织表达增高；AV-N3ICD组较空白组表达有极明显上升，表明腺病毒介导的N3ICD过表达成功。si-N3ICD组较空白组敲除效率接近50%，siRNA敲除效果理想。N3ICD 过表达一定程度上会提升实验细胞的凋亡比率，敲除则对凋亡比率没有明显影响。N3ICD 的敲除和过表达均在一定程度上降低细胞的增殖活性，并且N3ICD 过表达组降低程度更大。本研究表明Notch3信号通路在肺动脉高压中的重要作用，为探寻治疗新靶点提供依据。