重组人粒细胞集落刺激因子治疗大鼠肺动脉高压的效果及其对内皮祖细胞的影响

Effects of recombinant human granulocyte-colony stimulating factor therapy on rat pulmonaryhypertension and its influence on endothelial progenitor cells

目的探讨重组人粒细胞集落刺激因子（recombinant human granulocyte-colony stimulating factor, rhG-CSF）对大鼠肺动脉高压的治疗效果及其对循环内皮祖细胞（ endothelial progenitor cells, EPCs）数量及功能的影响。方法8周龄Sprague-Dawlay大鼠随机分为模型组、治疗组和对照组，每组8只。模型组及治疗组大鼠一次性皮下注射1％的野百合碱50mg／kg以诱导形成肺动脉高压模型，对照组注射等量磷酸盐缓冲液。治疗组于第5天皮下注射rhG-CSF50pg／（kg·d），连续3d。各组大鼠分别于第21天经尾静脉取血，应用流式细胞仪计数每100000个单个核细胞中EPCs所占比例。检测右心室收缩压，HE染色观察肺组织及肺血管病理变化。尾静脉采集的外周血单个核细胞进行EPCs体外培养，14d后采用四甲基偶氮唑蓝比色法、黏附实验和迁移实验测定EPCs的增殖、黏附和迁移能力。多组样本之间的比较采用单因素方差分析，组间两两比较采用LSD检验。结果（1）肺动脉压力及肺小动脉病理改变：模型组大鼠右心室收缩压较对照组明显升高[（48．13±2．85）mmHg与（27．88±3．04）mmHg，t=2．016，P〈0．01]，且肺小动脉血管内皮细胞损伤明显，管壁显著增厚。治疗组大鼠右心室收缩压[（30．38±2．83）mmHg]较模型组明显降低，接近对照组水平（t=0．376，P〉0．05），且肺部病变也相应减轻。（2）循环EPCs占单个核细胞比例：模型组大鼠外周血EPCs占单个核细胞比例较对照组明显下降[（0．016±0．007）％与（0．031±0．011）％，t=2．617，P〈0．01]。治疗后，大鼠循环EPCs占单个核细胞比例[（0．042±0．013）％]明显高于模型组（r=4．325，P〈0．01）及对照组（t=1．942，P〈0．05）。（3）体外培养的EPCs的增殖、黏附、迁移能力：模型组EPCs细胞增殖力、黏附细胞数和迁移细胞数分别为0．49±0．04，（6．93±1．47）个／高倍视野和（7．22±1．53）个／高倍视野，均低于对照组[分别为0．68±0．07、（11．05±1．73）个／高倍视野和（12．58±2．15）个／高倍视野]和治疗组[0．63±0．06、（12．35±1．82）个／高倍视野和（12．97±2．84）个／高倍视野]，差异均有统计学意义（P均〈0．05）。结论rhG-CSF能够有效上调肺动脉高压大鼠循环EPCs的数量及功能，从而有助于肺动脉高压的防治。

Objective To investigate the effects of recombinant human granulocyte-colony stimulating factor （rhG-CSF） therapy on pulmonary hypertension, and its influence on number and functions of circulating endothelial progenitor cells （EPCs） in rats. Methods Eight-week old Sprague-Dawlay rats were randomized into model group, treatment group and control group （8 rats in each group）. The rats in model group and treatment group were treated with single subcutaneous injection of 1% monocrotaline （50 mg/kg） to induce pulmonary hypertension models, while thecontrol group was lreated with phosphate buffered saline. Five days later, the rats in treatment group were administrated with 50 μg/（kg·d） rhG-CSF for 3 days. On day 21, peripheral blood was collected from caudal vein in all groups, and the percentage of EPCs in 100 000 mononuclear cells was evaluated by flow cytometry. Right ventricular systolic pressure was assessed, and the pathological changes of lung tissue and pneumoangiogram were observed by HE staining. Meanwhile, peripheral mononuclear cells collected from caudal vein were separated and cultured in vitro for EPCs. The cell functions as proliferation, adhesion and migration ability were assessed. ANOVA and LSD test were applied as statistical analysis methods. Results （1） The right ventricular systolic pressure of rats in model group was higher than that in the controls [（48.13±2. 85） mm Hg vs （27.88±3.04） mm Hg, t = 2.016, P〈0.01], the lesion of endothelial cells in pulmonary arteriolar was evident, and the vessel wall was thickened. The pulmonary artery pressure of rats in the treatment group [（30.38 ± 2.83） mm Hg] was lower than that in the model group and close to the level of control group （1= 0. 376,P〉0.05） with mild pulmonary pathological changes. （2） The percentage of peripheral blood EPCs in mononuclear cells in the model group was decreased as compared to the control group [（0. 016±0. 007）% vs （0.031±0.011） %, t=2.617, P〈0.01]. After administration ofrhG CSF, the EPCs in treatment group [（0. 042±0. 013）% ] was increased evidently as compared to the model group （t=4. 325, P〈0.01） and the control group （t=1. 942, P〈0.05）. （3） The proliferation, adhesive and migrated cells of EPCs in model group were 0.49±0.04,（6.93±1.47） cells/HPF and （7.22±1.53） cells/HPF,lower than those in control group [0.68±0.07,（11.05±1.73） cells/HPF and （12.58± 2.15） cells/HPF] and treatment group [0.63±0.06,（12.35±1.82） cells/HPF and （12.97±2.84） cells/HPF],the differences were statistically significant（all P〈0.05）. Conclusions rhG-CSF may be effective in treating pulmonary hypertension through up regulating the number and function of circulating EPCs in rat model of pulmonary hypertension.