脓毒症大鼠循环内皮祖细胞的数量变化及其意义

Change in number of circulating endothelial progenitor cells and its significance in septic rats

目的 观察并分析脓毒症时循环内皮祖细胞（cEPCs）数量变化及其意义.方法 采用盲肠结扎穿孔术制备雄性SD大鼠脓毒症模型（80只）,设正常对照组（16只）、假手术组（80只）.于制模后即刻、6、12、18 h及1、2、3、7 d各组取9只大鼠,动态观察外周血单个核细胞（PBMCs）中cEPCs数（流式细胞学法）及血中肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）、D-二聚体[酶联免疫吸附法（ELISA）]和抗凝血酶-Ⅲ（AT-Ⅲ,免疫浊度法）水平,肝、肾、肺组织湿/干重（W/D）比值.制模后1 d各组取8只大鼠,观察肝、肾、肺组织病理学变化及损伤评分.结果 制模后脓毒症大鼠cEPCs数量明显升高,于18 h达高峰[（7 161.9±689.8）个/106 PBMCs];血中TNF-α、IL-10、D-二聚体、AT-Ⅲ升高,分别于制模后12 h、12 h、3 d、18 h达峰值[（51.3±6.8） ng/L、（77.9±8.6） ng/L、（93.5±11.5） mg/L、（193.8±43.0） mg/L];肝、肾、肺W/D比值和组织损伤评分增加[18 h W/D比值：肝3.79±0.09,肾4.25±0.08,肺4.91±0.09;1 d组织损伤评分（分）：肝1.86±0.26,肾5.14±0.34,肺6.57±0.37].模型组上述各指标均显著高于同期假手术组[18 h cEPCs数量（2 235.5±472.7）个/106 PBMCs,12 h TNF-α （14.3±5.8） ng/L,12 h IL-10 （35.0±5.8） ng/L,3 d D-二聚体（14.2±4.4） mg/L,18 h AT-Ⅲ （100.1±12.8） mg/L;18 h W/D比值：肝3.50±0.07,肾3.96±0.04,肺4.54±0.14;1 d组织损伤评分（分）：肝0.29±0.18,肾0.57±0.20,肺1.14±0.51,P〈0.05或P〈0.01].相关分析显示,cEPCs数量与TNF-α（r=0.587）、IL-10（r=0.497）、D-二聚体（r=0.294）、AT-Ⅲ（r=0.690）及肝、肾、肺W/D比值（r1=0.532、r2=0.532、r3=0.679）均呈正相关（均P＜0.01）.结论 脓毒症时cEPCs数量明显升高,其变化与炎症反应、凝血激活、毛细血管渗漏和组织损伤程度呈正相关.提示cEPCs数量增加是机体对脓毒症的反应,且数量变化可能代表炎症反应和内皮与组织损伤的程度.

Objective To observe the change in number of circulating endothelial progenitor cells （cEPCs） and analyze its significance in septic rat. Methods Septic model of male Sprague-Dawley （SD） rats was reproduced by cecum ligation and puncture （n=80）, and the normal control group （n=16） and sham operation group （n=80） were established. Nine rats in each group were used, and the cEPCs numbers in peripheral blood mononuclear cells （PBMCs, by flow cytometry）, tumor necrosis factor-α （TNF-α）, interleukin-10 （IL-10）, D-dimer （by enzyme linked immunosorbent assay, ELISA）, antithrombase-Ⅲ （AT-Ⅲ, by immunonephelometry）, wet/dry （W/D） ratio of liver, kidney and lung were determined at 0, 6, 12, 18 hours and 1, 2, 3, 7 days after reproduction of model. Eight rats in each group were used, and the pathologic changes in liver, kidney and lung at 1 day were observed, and the injury scores were evaluated. Results The cEPCs number was markedly increased, reaching the peak [（7 161.9±689.8）/106 PBMCs] at 18 hours. Circulating TNF-α, IL-10, D-dimer, AT-Ⅲ were found to be increased, and the levels reached the peak at 12 hours, 12 hours, 3 days, 18 hours, respectively [（51.3±6.8） ng/L, （77.9±8.6） ng/L, （93.5±11.5） mg/L, （193.8±43.0） mg/L]. W/D ratio was elevated and signs of injury to the liver, kidney, lung became more obvious （18-hour W/D of liver： 3.79±0.09, kidney： 4.25±0.08, lung： 4.91±0.09; 1-day tissue evaluation of liver： 1.86±0.26, kidney： 5.14±0.34, lung： 6.57±0.37）. The levels of all parameters in model group were significantly higher than those in sham operation group [18-hour cEPCs numbers： （2 235.5±472.7）/106 PBMCs, 12-hour TNF-α： （14.3±5.8） ng/L, 12-hour IL-10： （35.0±5.8） ng/L, 3-day D-dimer： （14.2±4.4） mg/L, 18-hour AT-Ⅲ： （100.1±12.8） mg/L; 18-hour liver W/D ratio： 3.50±0.07, kidney： 3.96±0.04, lung： 4.54±0.14; 1-day tissue evaluation of liver： 0.29±0.18, kidney： 0.57±0.20, lung： 1.14±0.51, P〈0.05 or P〈0.01]. There was positive correlation between cEPCs numbers and TNF-α （r=0.587）, IL-10 （r=0.497）, D-dimer （r=0.294）, AT-Ⅲ （r=0.690）, and W/D ratio of liver, kidney, lung （r1=0.532, r2=0.532, r3=0.679, all P〈0.01）. Conclusion The cEPCs number markedly increases in septic rats, and it shows positive correlation with the degree of inflammatory reaction, blood clotting activation, capillary leakage and tissue damage. The increase of number of cEPCs is the result of reaction to sepsis, and its change in number might be valuable in evaluating the pathogenesis of sepsis.