脓毒症大鼠血清对内皮细胞的影响

Effect of serum from sepsis rats on human umbilical vein endothelial cells

目的研究脓毒症大鼠血清对人脐静脉内皮细胞(HUVEC)的影响,探讨脓毒症的发病机制。方法12只Sprague-Dawley大鼠随机分为假手术组和脓毒症组。采用盲肠结扎穿孔术制备大鼠脓毒症模型。术后6 h采血收集血清和血浆,采用鲎试剂法定量测定血浆脂多糖(LPS)水平,酶联免疫吸附试验测定血清肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6水平。并将血清与HUVEC孵育8 h后分别应用活细胞计数试剂盒(CCK-8)检测细胞活力,采用Hoechst 33342/碘化丙啶荧光染色计算细胞坏死率,并应用细胞划痕实验测定0、12、24 h的细胞间残存距离。结果脓毒症组LPS、TNF-α及IL-6水平均显著高于假手术组(P值均<0.01)。脓毒症组血清刺激HUVEC后8 h的细胞活力显著低于假手术组(P<0.01)。脓毒症组血清刺激HUVEC后的细胞坏死率显著高于假手术组(P<0.01)。孵育12和24 h后,脓毒症组的细胞间残存距离均显著大于假手术组(P值均<0.01)。结论脓毒症大鼠血清中LPS、细胞因子的水平显著升高,对血管内皮细胞有明显的杀伤抑制作用。

Objective To investigate the effect of serum of sepsis rats on human umbilical vein endothelial cell （HUVEC）, so as to probe into the pathogenesis sepsis. Methods Twelve Sprague-Dawley rats were randomly divided into sham-operation group （Sham） and sepsis group. Sepsis model was induced by cecal ligation and puncture with rats. Serum and plasma samples were collected 6 h after operation. The concentration of endotoxin in the plasma was determined by limulus test; the serum levels of tumor necrosis factor （TNF）-α and interleukin （IL）-6 were examined by enzyme linked immunosorbent assay （ELISA）. The HUVECs were incubated for 8 h in presence of the serum, and the cell counting kit （CCK）-8 assay was employed to test the cell viability. The Hoechst 33342 （HO）-propidiumiodide （PI） double fluorescent staining was used to calculate the necrosis rate of HUVECs, and the scratch test was used to examine the remaining distance between cells at 0 h, 12 h and 24 h. Results The CLP group had significantly higher levels of lipopolysaccharide （LPS）, TNF-α and IL-6 compared with the Sham-operation group （all P〈O. 01）. The HUVECs stimulated by serum of CLP group displayed significantly lower cell viability and higher necrosis rate compared with those stimulated by serum from the Sham- operation group （both P〈0. 0 1）. The remaining distance between cells was significantly greater in CLP group than that in the Sham-operation group at 12 h and 24 h （both P〈O. 01）. Conclusion The serum concentrations of ／ endotoxin and cytokines of sepsis rats are apparently elevated, and they have evident toxicity against endothelial cells. （Shanghai Med J, 2009, 32： 995-998）