HGF/c-met轴介导EPCs优势分布移植治疗缺氧性肺动脉高压

Endothelial cell injury and dysfunction is not only the initial step for the pulmonary arterial hypertension(PAH), also one of principal cause of adverse repair actions of pulmonary vascular injury. Transplantations of endothelial progenitor cell(EPCs) is the important therapy to the vascular injury, but deficiency of EPCs in recipients and no effective measures guiding to dominant distribution of pulmonary vascular injury restrict the curative effect and clinical implication of the treatment by transplantation of EPCs. Establishing the rat models with hypoxic PAH, with the methods of Liquid Scintillation Counting, MRI tracer, quantitative PCR, we collected the complete data of Cellular Localization and normal distribution of the transplanted EPCs. This program activated adequately EPCs in rats, with Isolation and Identification after cultivated in vitro, and constructed the adenovirus expression vector of c-met to transfect into EPCs in rats, then infused to the same rat. With the characteristic of increase of serum and hepatocyte growth factor (HGF) content in location of pulmonary vascular injury, we observe the influence and mechanism of HGF-c-met axes promotion the survival of transplanted EPCs and targeting distribution of PAH induced pulmonary vascular injury to repair, searching out further specificity and efficiency strategies for the treatment of PAH with EPCs transplantation to provide a new target of the prevention and cure of PAH. KEY WORDS: hypoxic pulmonary arterial hypertension; pulmonary vascular remodeling; HGF; dominant distribution

内皮细胞受损及功能障碍是肺动脉高压(PAH)的始动环节，也是肺血管损伤后 不良修复反应的主因之一。内皮祖细胞(EPCs)移植是治疗血管损伤的重要方法，但EPCs在受体内存活不足且尚无向损伤肺血管优势分布的有效措施，限制了EPCs移植治疗肺血管损伤应用及疗效。本项目以大鼠缺氧性PAH为模型，用液体闪烁计数、MRI示踪、定量PCR等方法获得移植的EPCs在大鼠体内的细胞定位和动态分布完整资料。充分动员大鼠自体EPCs，体外培养并分离鉴定，构建c-met的腺病毒表达载体，转染至EPCs，通过静脉回输同一鼠体内，利用肺血管损伤时血清及肺血管损伤部位肝细胞生长因子(HGF)含量增加的特点，观察 HGF/c-met轴促进自身EPCs及移植EPCs存活并向PAH损伤肺血管靶向分布修复肺血管损伤的影响及其可能机制，从整体-血管环-细胞三个层次寻求一种更为高效、特异的EPCs移植防治肺动脉高压的治疗策略。

肺动脉高压是一组由不同发病机制引起的以肺血管阻力持续增高、血管收缩、血管重建为特征的临床病理生理综合征。肺血管内皮损伤是肺动脉高压形成的始动因素。研究证实,内皮祖细胞可向血管受损部位聚集并参与血管修复和新生血管形成。肝细胞生长因子通过其受体c-met结合，促进内皮的修复，降低肺动脉压力。研究内容：观察c-Met-EPCs在移植体内的存活情况；获取c-Met-EPCs在大鼠体内的动态分布资料；观察HGF/c-met轴介导EPCs对损伤肺血管的收缩及重构的影响。方法：复制HPAH模型，原代培养EPCs并鉴定。构建带有c-Met基因的腺病毒并转染EPCs，细胞分为对照组、空载病毒组和c-Met过表达组。用Real-timePCR和Western blot检测c-Met mRNA和蛋白表达。用CM-Dil标记EPCs，通过尾静脉将EPCs输注到大鼠体内，观察EPCs在脏器中的分布。CCK8检测细胞增殖。Transwell 和Western blot检测细胞迁移能力和Akt、P-Akt的表达。将大鼠分为EPCs组、空载病毒组、c-met-EPCs组和正常大鼠共四组。尾静脉分别输注正常EPCs细胞、空载病毒转染的EPCs细胞、c-met-EPCs细胞和生理盐水。采集血清测定ET-1、NO的含量，右心导管法测定平均肺动脉压力。结果:c-Met-EPCs组中肺脏的细胞阳性率明显升高，在肾脏和脾脏的分布下降（P<0.001）。HGF在一定范围内呈浓度依赖性促进Ad-c-Met-EPCs迁移。HGF+Ad-c-Met-EPCs组的迁移能力和P-Akt的表达显著增高(P<0.05)。c-met-EPCs组与空载病毒组、EPCs组相比，ET-1显著降低（P<0.05）。c-met-EPCs组与空载病毒组、EPCs组相比，NO含量升高（P <0.05）。右心导管测定四组大鼠肺动脉压力，结果显示c-met-EPCs组与空载病毒组、EPCs组相比，平均肺动脉压(mPAP)降低（P<0.05）。结论:过表达c-Met蛋白促进EPCs优势分布于HPAH模型的肺组织。HGF/c-Met能够显著提高EPCs的迁移能力；HGF/c-Met可能通过PI3K/Akt信号通路促进EPCs的迁移。HGF/c-Met能优势分布于损伤的肺动脉，修复受损的肺血管,使肺动脉压力下降，NO含量升高，ET-1含量下降。