高糖培养脂多糖刺激下肺脏微血管内皮细胞通透性改变及DDAH／NOS／NO失平衡在其发生机制中的作用  

High glucose enhances permeability of human pulmonary microvascular endothelial cells by lipopolysaccharide stimulation in vitro and effect of DDAH/NOSfNO imbalance on its pathogenesis

作　　者：刘秀娟[1] 穆恩[1] 梁英健[1] 章志丹[1] 马晓春[1] 

机构地区：[1]中国医科大学附属第一医院重症医学科,辽宁沈阳110001

出　　处：《中华危重病急救医学》2013年第3期140-144,共5页Chinese Critical Care Medicine

基　　金：基金项目：高等学校博士学科点专项科研基金资助项目（20102104110003）

摘　　要：目的探讨高糖对脂多糖（LPS）刺激下血管内皮细胞损伤的影响及其机制。方法将人肺脏微血管内皮细胞（PMVEC）分为正常糖组（NG组）、正常糖＋LPS刺激组（NGL组）、高糖组（HG组）、高糖＋LPS刺激组（HGL组），分别给予含10％小牛血清的正常糖（5．5mmol／L）或高糖（33mmol／L）培养5d，加入10mg／LLPS刺激细胞24h。采用免疫荧光染色观察细胞纤维肌动蛋白（F-actin）的分布及变化；扫描电镜观察细胞膜窗孔数量及孔径变化；细胞迁移实验（Transwell）测定单层内皮细胞的辣根过氧化物酶（HRP）通透性；硝酸盐还原法（Griess法）检测细胞培养上清液中一氧化氮（NO）含量；蛋白质免疫印迹试验（Western blotting）测定细胞二甲基精氨酸-二甲胺水解酶2（DDAH2）、诱生型一氧化氮合酶（iNOS）、内皮型一氧化氮合酶（eNOS）的蛋白表达。结果与NGL组比较，HGL组PMVEC的F-actin分布排列紊乱，细胞膜窗孔异常增大、增多，细胞单层对HRP通透率增加[（53．62±6．70）％比（23．63±3．92）％，P〈0．01]，细胞DDAH2表达（积分A值）减少（0．33±0．08比0．77±0．14，P〈0．01），iNOS表达（积分A值）增加（1．40±0．29比1．04±0．09，P〈0．01），eNOS表达（积分A值）减少（0．67±0．09比0．91±0．17，P〈0．05），上清液NO含量（μmol／L）增多（20．36±2．25比7．99±0．33，P〈0．01）。结论高糖加重LPS刺激下体外培养PMVEC的F—actin分布紊乱、单层细胞通透性增加；NO调节紊乱可能参与了PMVEC损害的发生。Objective To investigate the damage to endothelial cells incubated in high concentration of glucose challenged by lipopolysaccharide （LPS）, and the likely mechanisms of injury. Methods Human pulmonary microvascular endothelial cells （PMVECs） were divided into the following groups： normal glucose group （NG）, normal glucose ± LPS stimulation group （NGL）, high glucose stimulation group （HG）, and high glucose ± LPS stimulation group （HGL）. The cells were incubated with normal glucose （5.5 mmol/L, contained 10% calf serum） or high glucose （33 mmol/L） for 5 days to form a monolayer of cells before LPS stimulation （ 10 mg/L） for 24 hours. The mierofilaments （F-actin） were investigated by immuno-fluorescence, and the number and size change in fenestrae were examined by scanning electron microscopy. The permeability of vascular endothelial cell was assessed by trans-PMVEC horseradish peroxidase （HRP） flux. Western blotting was used to determine the expressions of dimethylarginine dimethylaminohydrolase 2 （DDAH2）, inducible nitricoxide synthase （iNOS） and endothelial nitricoxide synthase （eNOS）. Nitric oxide （NO） was assessed by Griess method. Results When stimulated with LPS, cells incubated with high glucose showed obvious microfilament rearrangement, a larger average diameter and increased number of F-aetin, as well as higher HRP permeability on the hyperglycemic PMVECs compared with PMVECs cultured with normal glucose （53.62 ± 6.70）% vs. （23.63 ± 3.92）%, P〈0.01]. Furthermore, high glucose down-regulated DDAH2 expression （arbitrary units, AU, 0.33 ± 0.08 vs. 0.77 ± 0.14 , P〈0.01）] and up-regulated LPS-stimulated iNOS production （ 1.40 ± 0.29 vs. 1.04 ± 0.09, P〈0.01 ）, as well as increased LPS-stimulated nitrite/nitrate and stable NO end products （μmol/L） compared with normal （20.36 ± 2.25 vs. 7.99 ± 0.33, P〈0.01 ） and reduction of eNOS levels was observed （0.67 ±0.09 vs. 0.91 ±0.17, P〈0.05）. Conclusion It de

关 键 词：高糖 内毒素 二甲基精氨酸-二甲胺水解酶2 一氧化氮合酶 内皮细胞通透性 

分 类 号：R363[医药卫生—病理学]