血内皮祖细胞与一氧化氮在慢性阻塞性肺疾病并发肺动脉高压患者中的变化及意义

Changes of endothelial progenitor cells and nitric oxide in patients with pulmonary arterial hypertension secondary to chronic obstructive pulmonary disease

目的 观察慢性阻塞性肺疾病（COPD）并发肺动脉高压（PAH）病人外周血内皮祖细胞（EPCs）数量和功能及一氧化氮的变化,并探讨其在COPD并PAH发病中的作用.方法 选择本院住院的COPD病人60例,其中COPD并PAH（COPD＋PAH组）病人30例,COPD非PAH病人30例（COPD组）;正常对照20例（非COPD组）.用密度梯度离心法从外周血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板,培养7d后对贴壁细胞进行细胞化学分析.通过集落形成试验、改良的Boyden小室和黏附能力测定实验计数EPC的数量、测定EPC的迁移和黏附能力.用硝酸还原酶法比色检测入选患者血浆一氧化氮（NO）水平.结果 和正常对照组[（24.9±4.1）克隆形成数]及COPD组[（21.9±3.9）克隆形成数]相比,COPD＋PAH组[（14.2±3.5）克隆形成数]外周血EPC数量明显减少,黏附和迁移能力显著降低（F≥9.15,q≥3.49,P〈0.05）,且EPC数量及黏附、迁移能力与肺动脉压力呈负相关（r=-0.79、-0.85、-0.89,P〈0.01）.COPD＋PAH组患者NO水平[（43.6±4.8）ng/ml]明显低于对照组[（67.17±4.9）ng/ml]（P〈0.01）,NO浓度与循环EPCs数量及迁移能力呈正相关（r=0.77、0.71,P〈0.01）,与循环EPCs黏附能力无关.结论 COPD病人PAH的发生可能与循环EPCs数量减少、迁移和黏附能力降低有关,这种变化可能与血浆NO水平减少有关.

Objective To investigate the changing of endothelial progenitor cells and nitric oxide in patients with pulmonary arterial hypertension secondary to chronic obstructive pulmonary disease.Method Mononuclear cells from 20 healthy persons （the control group, n = 20） , COPD patients without PAH （COPD non-PAH group, n =30） and patients with PAH secondary to COPD （COPD and PAH group, n=30） were investigated. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation. EPC number and migration were assayed by colony forming unit-EPCs （CFU-EPCs） assay and modified Boyden chamber assay , respectively. EPC adhesion assay was performed by replanting those on fibronectin-coated dishes , then adherent cells were counted. The concentration of NO was measured with method of nitrate reductase.Result The numbers of CFU and migration, adhesion activity of circulating EPCs in COPD[ （21.9±3. 9）CFU-EPC] and PAH group[ （14. 2 ±3. 5）CFU-EPC] were significantly lower than that in non-COPD group and COPD non-PAH group [ （ 24.9 ±4.1） CFU-EPC ]. It was also observed that a strong negative correlation between the levels of PAH and the numbers of CFU and adhesion, migration activity of circulating EPCs. The NO level in the PAH group [ （43. 6 ±4. 8）ng/ml] was significantly lower than that in control group [ （ 67. 17±4.9 ） ng/ml ] （ P 〈 0.01）. The NO level was positively correlated with number and migration ability of EPCs（ r =0. 77,0.71, P 〈0.01） , but not correlated with adhesion ability.Conclusion The number of CFU and migratory, adhesive activity of EPC in patients withPAH secondary to COPD was significantly decreased. These changes may be associated with low level of plasma NO.