SIRT1调节eNOS-NO通路在慢性血栓栓塞性肺动脉高压血管重塑中的作用机制

Chronic thromboembolism pulmonary hypertension (CTEPH) is a fatal complication with the predisposing factor of recurrent pulmonary embolism (PE). CTEPH is significantly characterized by pulmonary vascular remodeling with the key cellular basis of endothelial cells apoptosis and smooth muscle cells proliferation. Further insight of CTEPH pathogenesis is of important significances to new target in prevention and treatment of pulmonary hypertension. It has been suggested that the elevation of intracellular calcium concentration[Ca2+]i can promote the generation of NO (nitric oxide) activated by the key enzyme eNOS (endothelial nitric oxide synthase) and participate in the protection of vascular function. With the same function, Resveratrol, a SIRTl (silent information regulator 2-related enzymes 1) activator can inhibit the proliferation of vascular smooth muscle as well. On these grounds, we gain hypothesis: up-regulated expression of the SIRT1 by Resveratrol regulates eNOS-NO system with the change of intracellular calcium concentration, which may play a protective role of vascular remodeling in occurrence and development of CTEPH. This study will focus on the pulmonary vascular remodeling which is the critical point of CTEPH development and prognosis. We aim to explore the mechanism of SIRT1 in inhibiting vascular smooth muscle proliferation and improving endothelial dysfunction in the process of CTEPH vascular remodeling by the construction of Resveratrol and interference system at the cellular level, establishment of animal model to study the mechanism and interaction between Resveratrol and eNOS-NO at the model level. Our research will be helpful in seeking for interventions targeting and providing new ideas for treatment and the development of new drugs for CTEPH.

慢性血栓栓塞性肺动脉高压（CTEPH）是一种以肺血管重塑为显著特点的致死性疾病。肺栓塞反复发作是其高危易患因素；内皮细胞凋亡，平滑肌细胞增殖是肺血管重塑的关键细胞学基础，深入探讨发病机制对寻找有效的肺动脉高压治疗方法有重要意义。研究证实当细胞内钙离子浓度升高时，利于NO合成限速酶eNOS的激活，促进NO生成，对血管内皮起到保护作用；去乙酰化酶SIRT1有同样作用；SIRTl的激活剂白藜芦醇（Resveratrol）可以抑制血管平滑肌的增殖。因此我们提出假说：Resveratrol上调SIRT1表达，通过调节[Ca2+]i变化影响eNOS-NO系统，在CTEPH血管重塑的发生发展中起保护性作用。本研究着眼于肺血管重塑这一影响CTEPH发展和预后关键点，在细胞水平通过建立过表达及基因干扰体系，利用多种研究手段探讨SIRT1在抑制CTEPH血管重塑过程中血管平滑肌增殖和改善内皮功能紊乱中的作用，并建立动物模型，从整体水平探讨Resveratrol作用机制以及与eNOS-NO的相互作用，寻找针对CTEPH患者有诊治价值的干预靶点，为CTEPH的治疗及新药开发提供新思路。

慢性血栓栓塞性肺动脉高压（CTEPH）发病源于不溶的肺血栓栓塞及继发的肺血管重塑，并最终导致肺动脉高压、右心衰竭，深入认识CTEPH患者肺动脉高压的细胞和分子机制，明确其肺血管重塑的发生发展机制，对CTEPH的预防、治疗和降低死亡率具有积极现实意义。.本项目着眼于肺血管重塑这一影响CTEPH发展和预后的关键环节，合理利用临床标本构建细胞模型，并建立动物模型，运用细胞及分子生物学多种实验方法从基因表达调控和蛋白表达方面，对SIRT1抑制CTEPH血管重塑过程中血管平滑肌增殖作用及其与eNOS的相互关系进行研究。.本项目利用体外培养的CTEPH患者肺动脉血栓内膜剥脱标本中的PASMCs为细胞模型，明确SIRT1在PASMCs中的表达情况，并与正常肺动脉平滑肌细胞系（HPASMC）中其mRNA含量进行比较，以及相应eNOS的表达变化情况，结果提示SIRT1及eNOS表达均有升高。检测正常对照及CTEPH患者外周血中SIRT1及eNOS mRNA水平表达情况及相应血清中浓度变化，与正常对照比较，提示CTEPH患者外周血SIRT1及eNOS mRNA水平和血清中浓度均有明显降低。对固定的肺动脉内膜剥脱术后组织标本行免疫组化染色观察SIRT1及eNOS的组织分布，与正常对照比较，提示肺组织内SIRT1及eNOS表达均有降低。.本项目成功建立CTEPH小鼠模型，不断优化建模方法，进一步提高模型的稳定性，从整体水平构建SIRT1和eNOS-NO的协同作用网络。此部分小鼠模型制备结果已整理完毕，准备投稿。.本项目组紧密跟随精准医学发展，已开展组学相关实验。充分合理利用外周血标本，进行基因芯片检测，研究mRNA，microRNA和circRNA的表达变化，以期构建CTEPH发生的RNA调控网络，并通过多种生物信息学分析方法，筛选出差异特征显著基因，为后续开展靶基因预测及相关功能验证提供实验基础，也为有效诊治疾病、寻找潜在生物标志物和药物靶点提供必要理论基础。此部分内容已写成文章，准备投稿。.以上结果均为SIRT1及eNOS作为针对CTEPH患者有预测和诊断价值的干预靶点提供实验依据，并为明确其作为CTEPH诊断和治疗监测中有潜在临床应用价值的血清学标志物提供重要实验基础。