lipid raft/caveolae调节PMVECs接触性抑制在肝肺综合症肺微血管扩张中的作用和机制研究

Hepatopulmonary syndrome (HPS) is a pulmonary vascular complication caused by chronic hepatosis,a key pathophysiological component of which is IPDV.Our previous study comfirmed that the abnormal proliferation and myogenic differentiation of PMVECs aggravated the progression of IPVD.We found that HPS rat serum stimulated PMVECs stratified growth from signal-layer,and pulmonary microvascular wall of HPS rat thickened, which could not be explained by proliferation and differentiation and indicate an obvious attenuation of contact inhibition and may become a new target of HPS treatment. Since lipid raft/cavelae play an important role in contact inhibiton, we plan to structure in vivo and in vitro HPS model to observe the expressions of contact inhibtion-related proteins in lipid raft/caveolae of PMVECs;construct Ad-intergrin vectors,disrupt lipid raft and apply siRNA to knockdown caveolin to detect the changes of microvascular wall and related protein expression. Therefore, elucidate the role of lipid raft/caveolae regulating contact inhibition in IPVD of HPS，and provide a new insight in prevention and treatment of HPS.

肝肺综合症（HPS）是由慢性肝病引起的肺血管并发症,其主要病理变化特征是肺内微血管扩张（IPVD），我们的前期研究证实肺微血管内皮细胞（PMVECs）发生异常增殖和肌样分化,从而加重IPVD进展。研究中我们发现HPS大鼠血清刺激PMVECs由单层向复层生长，HPS大鼠肺微血管管壁增厚，难以用增殖和分化解释，提示可能存在明显的接触性抑制减弱，有望成为HPS防治的新靶点。鉴于lipid raft/caveolae在接触性抑制中起重要作用，本课题拟建立在体和离体模型，观察HPS时PMVECs中lipid raft/caveolae接触性抑制相关蛋白表达变化；构建Ad-intergrin载体，破坏脂质筏，siRNA技术干预caveolin，检测肺微血管管壁变化及上述蛋白表达变化，以阐明lipid raft/caveolae 调节细胞接触性抑制在HPS中IPVD中的作用，为HPS的防治提供新思路.

肝肺综合症（HPS）是由慢性肝病引起的肺血管并发症,其主要病理变化特征是肺内微血管扩张（IPVD），我们的前期研究证实肺微血管内皮细胞（PMVECs）发生异常增殖和肌样分化,从而加重IPVD 进展。鉴于lipid raft/caveolae在多种肿瘤细胞接触性抑制失活导致的异常增殖中起重要作用，本项目以lipid raft/caveolae为核心，研究细胞接触性抑制失活在HPS的IPVD行程中的作用，并探索其上游调控机制。本项目通过在体和细胞实验，采用RT-PCR、western blotting、MTT、TUNEL、免疫荧光、组织切片分析、动脉血气分析等技术研究发现：①HPS大鼠血清降低PMVECs中miR-199a-5p的表达，通过负调控使Cav-1的表达增加。②Cav-1 siRNA导致PMVECs中应力纤维的丢失和Rac1激活，从而减弱miR-199a-5p介导的PMVECs增殖。③Annexin A2通过下调paxillin的表达抑制HPS大鼠血清诱导的PASMCs迁移功能增强。④水通道蛋白1（aquaporin 1）通过p38-MAPK信号通路促进HPS血清刺激诱导的PASMCs迁移功能增强。⑤抑制自噬水平可减轻HPS大鼠肺微血管扩张和内皮细胞异常增生。上述结果证实CBDL大鼠血清诱导miR-199a-5p表达减少，通过负调控使Cav-1的表达升高进而使应力纤维形成增多促进PMVECs异常增殖。调控miR-199a-5p/ Cav-1/Rac1信号通路可以明显抑制PMVECs的异常增殖，可能成为HPS治疗的重要靶点。同时我们还发现AQP-1和Cav-1下游ANXA2在调控HPS血清致PASMCs迁移能力增强上起到重要作用。动物试验中我们发现HPS大鼠肺组织早期自噬增加从而促进了疾病发展后期肺内血管扩张（IPVD）和PMVECs异常增殖的病理过程，进一步阐明了CBDL大鼠模型HPS发生的机制，为更好的防治HPS提供了新的思路。