线粒体动力学在不可逆肺血管病变形成过程中的作用

The irreversible pulmonary vascular lesions are the primary cause of incurability of severe pulmonary hypertension. These lesions characterized by apoptosis resistance in pathological change, which are similar to that in tumor cells, while mitochondrial dysfunction is an important pathophysiological mechanism of tumor, which is also involved in the regulation of pulmonary artery smooth muscle cell proliferation in pulmonary vascular remodeling. Endothelial cells play a more important role than pulmonary artery smooth muscle cell in the pulmonary remodeling, and mitochondrial dynamics is the basis of its physiological activity. Therefore, the present study aims to identify the effect of mitochondrial dynamics in the formation of irreversible pulmonary remodeling by detection of mitochondrial fission/fusion and the expression of regulatory protein for it including Mfn1, Mfn2, OPA1, drp1 in pulmonary endothelial cells from reversible and irreversible pulmonary vascular disease model, and investigation of the proliferation and apoptosis of pulmonary endothelial cells. Moreover, the effect of mitochondrial dynamics are identified by the application of an inhibitor of drp1. The effects of mitochondrial dynamics on the apoptosis of endothelial cells in the irreversible pulmonary remodeling are also explored by cell experiment. The physiopathologic processes of endothelial cell in the pulmonary remodeling is imitated by culture of apoptosis endothelial cells induced by TNF-αin CELLMAX system with high shear stress, then the mitochondrial fission/fusion, the expression of Mfn1、Mfn2、OPA1、Drp1 are investigated in these endothelial cells with apoptosis resistance. The effects of mitochondrial dynamics are evidenced by the change of proliferation and apoptosis of endothelial cell with defect of Mfn2 or drp1 knocked out by CRISPR/cas9. The mechanisms of mitochondrial dynamics on endothelial apoptosis are explored by knocking out its downstream effectors including Kv1.5 channel and cytochrome c. In summary, the present study is to illuminate the possible mechanisms of development of apoptosis resistance in endothelial cells from apoptosis, and provide a theoretical basis for PAH targeted drug development.

肺血管不可逆病变是肺动脉高压无法治愈的根本原因，凋亡抵抗是其典型病理特征，与肿瘤细胞相似。线粒体异常是肿瘤的重要病理生理机制，也参与调控肺动脉平滑肌细胞异常增殖，而内皮细胞在肺血管重构过程中起更关键作用。线粒体动力学则是自身生理活动的基础。本研究通过可逆、不可逆肺血管病变模型，检测肺动脉内皮细胞线粒体融合/分裂和调节蛋白Mfn1、Mfn2、OPA1、Drp1变化，肺动脉内皮细胞增殖/凋亡变化，并Drp1抑制剂干预；然后，细胞学分析不可逆病变内皮细胞凋亡与线粒体动力学的关系；并经TNF-α诱导内皮细胞凋亡后，高剪切应力CELLMAX培养系统中诱导凋亡抵抗，模拟内皮细胞的病理生理过程，CRISPR/Cas9敲除Mfn2，Drp1，明确线粒体动力学与内皮细胞增殖/凋亡的关系；敲除下游Kv1.5通道和cytc完善其机制。阐明内皮细胞从凋亡发展到凋亡抵亡抵抗的可能机制，为靶向药物开发提供理论依据。

肺动脉高压是肺血管重构为病理特征的致死性疾病，以细胞凋亡抵抗为特征的丛样病变是其终末期的表现。由于病理特征的相似性，肿瘤细胞中发生的线粒体异常在肺血管重构过程中的作用也引起了关注，尤其是代谢异常；线粒体在融合/分裂之间的动态平衡是维持生物体众多生理活动的基础，也是调节其自身功能的基础，包括代谢、凋亡等。本研究结合了动物和细胞学实验，发现线粒体异常分裂参与了肺动脉内皮细胞细胞凋亡抵抗的发生，导致最终的肺动脉丛样病变，Drp1在这一过程中起重要的调节作用，其抑制剂Mdivi-1可抑制肺动脉丛样病变的形成；而原代培养的丛样病变来源内皮细胞和TNF-α+高剪切应力诱导的内皮细胞凋亡抵抗模型则再次证实了Drp-1调控的线粒体分裂在内皮细胞从凋亡发展到凋亡抵抗过程中的作用，基因敲除Drp1阻止了内皮细胞凋亡抵抗的发生。Mfn2、Cyt C、Kv1.5等线粒体调节蛋白也可能参与内皮细胞凋亡抵抗，而STAT3磷酸化激活是Drp1过表达的可能原因。本研究从线粒体动态平衡的角度，在亚细胞水平探索了肺血管丛样病变的病理机制，加深了肺动脉高压发病机制的理解，为肺动脉高压的治疗开辟了新的方向。