中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2013年
3期
140-144
,共5页
刘秀娟%穆恩%梁英健%章志丹%马晓春
劉秀娟%穆恩%樑英健%章誌丹%馬曉春
류수연%목은%량영건%장지단%마효춘
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
高糖%內毒素%二甲基精氨痠-二甲胺水解酶2%一氧化氮閤酶%內皮細胞通透性
고당%내독소%이갑기정안산-이갑알수해매2%일양화담합매%내피세포통투성
High glucose%Endotoxin%Dimethylarginine dimethylaminohydrolase 2%Nitricoxide synthase%Endothelial cell permeability
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(5.5 mmol/L)或高糖(33 mmol/L)培养5d,加入10 mg/L LPS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.
目的 探討高糖對脂多糖(LPS)刺激下血管內皮細胞損傷的影響及其機製.方法 將人肺髒微血管內皮細胞(PMVEC)分為正常糖組(NG組)、正常糖+LPS刺激組(NGL組)、高糖組(HG組)、高糖+LPS刺激組(HGL組),分彆給予含10％小牛血清的正常糖(5.5 mmol/L)或高糖(33 mmol/L)培養5d,加入10 mg/L LPS刺激細胞24h.採用免疫熒光染色觀察細胞纖維肌動蛋白(F-actin)的分佈及變化；掃描電鏡觀察細胞膜窗孔數量及孔徑變化；細胞遷移實驗(Transwell)測定單層內皮細胞的辣根過氧化物酶(HRP)通透性；硝痠鹽還原法(Griess法)檢測細胞培養上清液中一氧化氮(NO)含量；蛋白質免疫印跡試驗(Western blotting)測定細胞二甲基精氨痠-二甲胺水解酶2(DDAH2)、誘生型一氧化氮閤酶(iNOS)、內皮型一氧化氮閤酶(eNOS)的蛋白錶達.結果 與NGL組比較,HGL組PMVEC的F-actin分佈排列紊亂,細胞膜窗孔異常增大、增多,細胞單層對HRP通透率增加[(53.62±6.70)％比(23.63±3.92)％,P＜0.01],細胞DDAH2錶達(積分A值)減少(0.33±0.08比0.77±0.14,P＜0.01),iNOS錶達(積分A值)增加(1.40±0.29比1.04±0.09,P＜0.01),eNOS錶達(積分A值)減少(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).結論 高糖加重LPS刺激下體外培養PMVEC的F-actin分佈紊亂、單層細胞通透性增加；NO調節紊亂可能參與瞭PMVEC損害的髮生.
목적 탐토고당대지다당(LPS)자격하혈관내피세포손상적영향급기궤제.방법 장인폐장미혈관내피세포(PMVEC)분위정상당조(NG조)、정상당+LPS자격조(NGL조)、고당조(HG조)、고당+LPS자격조(HGL조),분별급여함10％소우혈청적정상당(5.5 mmol/L)혹고당(33 mmol/L)배양5d,가입10 mg/L LPS자격세포24h.채용면역형광염색관찰세포섬유기동단백(F-actin)적분포급변화；소묘전경관찰세포막창공수량급공경변화；세포천이실험(Transwell)측정단층내피세포적랄근과양화물매(HRP)통투성；초산염환원법(Griess법)검측세포배양상청액중일양화담(NO)함량；단백질면역인적시험(Western blotting)측정세포이갑기정안산-이갑알수해매2(DDAH2)、유생형일양화담합매(iNOS)、내피형일양화담합매(eNOS)적단백표체.결과 여NGL조비교,HGL조PMVEC적F-actin분포배렬문란,세포막창공이상증대、증다,세포단층대HRP통투솔증가[(53.62±6.70)％비(23.63±3.92)％,P＜0.01],세포DDAH2표체(적분A치)감소(0.33±0.08비0.77±0.14,P＜0.01),iNOS표체(적분A치)증가(1.40±0.29비1.04±0.09,P＜0.01),eNOS표체(적분A치)감소(0.67±0.09비0.91±0.17,P＜0.05),상청액NO함량(μmol/L)증다(20.36±2.25비7.99±0.33,P＜0.01).결론 고당가중LPS자격하체외배양PMVEC적F-actin분포문란、단층세포통투성증가；NO조절문란가능삼여료PMVEC손해적발생.
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 microfilaments (F-actin) were investigated by immuno-fluorescence,and the number and size change in fenestme 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-actin,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 demonstrated that,in vitro high glucose deteriorate LPS-stimulated F-actin rearrangement and hyperpermeability of an endothelial monolayer,and the worsened imbalance of the NO pathway may lead to endothelial damage in microcirculation.
