肾上腺素受体β2对COPD患者外周血中EPCs修复功能影响及机制研究

. It is accepted in respiratory field that COPD patients were two to four times more likely to die of cardiovascular disease at a 3-year follow-up than were age- and sex-matched control subjects.Greater than 44% of these patients present with generalized atherosclerosis at autopsy. Circulating endothelial progenitor cells (EPCs) can restore injured endothelium and thereby protect against atherosclerotic vascular disease.In our study,we found that the EPCs capacities of migration ,adhesion to injured sites were dysfunctional in COPD patients.whether the function altered were relation with abnormal expression of β2AR in EPCs from COPD patients was unclear. In our study,the expression of β2AR was observed through FACS,RT-PCT and Westernblot.The altered expression of β2AR led to dysfunction of Epics,including proliferation,differentiation,adheresion,chemoattractive etc.The dysfunction of COPD patients EPCs in perpherial blood was improved by down-regulated expression of β2AR through si-RNA technology. The effect of β2AR on function of EPCs may be affected by PKA/ERK1/2 signal actived.The antagonist or agonist of PKA/ERK1/2 were used to study the changed function of COPD patients EPCs. In vivo,EPCs from COPD patients were transfected by si-RNA and injected into injured femoral artery of NOD/SCID.The capacity of adhere to injured sites was determined by slice fluorescence immunostained.In this study,our aim is to determine if and how the expression altered of β2AR in EPCs are involved in the EPCs dysfunction and vascular repair process in COPD patients.

慢性阻塞性肺病患者心血管患病率较同等条件下对照人群高，这一论断已被呼吸界认同。研究表明外周血中表达EPCs参与受损血管的修复，本课题在前期研究中发现慢性阻塞性肺病患者外周血中表达EPCs修复功能障碍，是否与其β2AR表达异常有关尚不清楚。本课题应用流式、RT-PCR及Westernblot技术,观察患者外周血中EPCsβ2AR表达；通过基因干扰技术上调或下调β2AR表达，观察对EPCs增殖、分化、趋化、粘附的影响。通过对β2AR信号通路PKA/ERK1/2的阻断或激动，观察EPCs上述细胞功能的变化，揭示β2AR对患者EPCs功能影响的机制；将经过转染si-β2AR后的患者外周血中EPCs注入下肢股动脉内皮损伤SCID小鼠模型体内，进一步观察β2AR对EPCs修复功能的影响。

WHO在2007年调查预测到2030年因死亡人数将升到各种疾病死亡第四位。许多资料表明COPD与心血管疾病的发生存在相关性，COPD成为心血管疾病发生的独立危险因子。EPCs参与受损处血管内皮细胞修复，实验证明2%～30%受损血管内皮是由EPCs分化而来，动物模型表明在受损处EPCs参5%～25%新生血管的形成。外周血中EPCs数量的多少对修复功能正常发挥起着重要作用。临床资料表明外周血中EPCs数量降低可造成心绞痛频率和其他心血管事件发生率增加。提示外周血液中EPCs数量降低，影响受损血管的修复，造成重要血管功能障碍，诱发患者一系列心血管症状。.很多资料表明：由于EPCs数量及功能降低造成糖尿病患者血管闭塞性疾病的发生，同时也是引起重肺动脉高压的重要因素，我们推断COPD患者外周血中EPCs数量及功能的降低可能是引起其心血管疾病发生的重要原因。功能改变可能与EPCs上β2AR、CXCR4的表达与受体活性有关，干扰EPCs向外周血中迁移，引起外周血中EPCs细胞黏附、分化、血管形成等细胞功能异常，并进一步造成外周血中细胞数量降低，使受损血管内皮的修复障碍，导致血管内皮功能异常，加速部分动脉硬化进程，促进COPD患者心血管疾病的发生。.主要内容：.1通过CD34+正分选试剂盒进行磁珠纯化，观察中、重度COPD患者外周血中CD34+细胞数量变化。.2通过对来自COPD患者外周血EPCs体外培养，观察β2AR表达情况。.3应用相应β2AR激动剂或拮抗剂，观察EPCs体外增殖、趋化、黏附及血管形成等细胞功能的变化。.4在上述研究的基础上，应用阻滞剂或激动剂作用于PKA/ERK1通路，观察COPD患者EPCs体外增殖、趋化、黏附及成血管等功能的变化情况。.结果：1、COPD患者组与对照组外周血中的CD34+细胞数明显加少。增殖、迁移CXCR4表达均降低。COPD患者的late-EPC小管黏附能力降低，不能形成完整的小管。.3、COPD患者β2AR蛋白表达强度高于对照组细胞。肾上腺素受体β2抑制剂及抗体可不同程度提高COPD患者early-EPCs增殖、迁移能力；肾上腺素受体β2激动剂则相反；在COPD患者early-EPCs上，肾上腺素受体β2表达增高，导致CXCR4表达降低，增加细胞内ROS水平。.4、没有观察到PKA/ERK1通路对EPCs功能的影响。

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