摘要
目的从剂量~效应和时间一效应关系探讨脂多糖(LPS)对大鼠主动脉内皮细胞(RAECs)血管性血友病因子裂解酶(ADAMTS-13)mRNA和蛋白表达的影响。方法采用组织贴块法培养Wistar大鼠的RAECs,1周后按1:3传代至第4~5代,将细胞分为空白对照组和0.01、0.1、1、5μg/mlLPS刺激组,分别于12、24、48、72h用半定量逆转录-聚合酶链反应(RT—PCR)测定内皮细胞ADAMTS-13mRNA表达;用酶联免疫吸附法(ELIsA)检测上清液内ADAMTS-13蛋白水平。结果空白对照组有一定量的ADAMTS~13mRNA和蛋白表达。随着LPS刺激浓度增加和刺激时间延长,ADAMTS-13mRNA和蛋白表达逐渐下降。与空白对照组(25.22±1.41)比较,0.01μg/mlLPS刺激48h时ADAMTS-13mRNA表达(18.78±0.86)即明显下降(P〈0.01);0.1μg/ml和1μg/ml LPS刺激24h时ADAMTS-13 mTIRNA表达(23.43±0.63、22.41±0.76)即明显下降(P〈0.05和P〈0.01);5μg/ml LPS刺激12h时ADAMTS-13 mRNA表达(20.01±2.47)即明显下降(P〈0.01)。与空白对照组[(115.76±2.36)ng/m13比较,0.01μg/mlLPS刺激12h时ADAMTS-13蛋白表达[(113.43±1.07)ng/m13即明显下降(P〈0.05);至5ug/ml LPS刺激72h时ADAMTS-13蛋白表达[(7.63±2.64)ng/mB降至最低(P〈0.01)。结论RAECs有一定量的ADAMTS-13mRNA和蛋白表达;不同浓度LPS刺激内皮细胞不同时间后ADAMTS-13mRNA和蛋白表达降低,具有剂量依赖性和时间依赖性。
Objective To investigate the dose-response effect and time-response effect of lipopolysaccharide (LPS) on yon Willebrand factor cleaving protease (ADAMTS-13, vWF-cp) mRNA expression and protein in rat aortic endothelial cells (RAECs). Methods RAECs were grown by culturing of aortic tissue. When ARECs were cultured for one week, it was co-cultured by 1 : 3 to reach 4- 5 generations. ARECs were randomly divided into five groups: control group and four LPS stimulation groups (0.01, 0. 1, 1 and 5 μg/ml). The RAECs and supernatants were obtained at 12, 24, 48, and 72 hours after being stimulated by LPS. ADAMTS-13 mRNA expression of RAECs was assessed by quantitation reverse transcription-polymerase chain reaction (RT-PCR), and protein of ADAMTS-13 in supernatants was determined by enzyme-linked immunoadsorbent assay (ELISA). Results In the control group RAECs were shown to express ADAMTS-13 at both protein and mRNA levels. With the increase of concentration of LPS, or increase in stimulus duration, expression of ADAMTS-13 mRNA and protein were gradually lowered. Compared with the control group (25.22 ± 1.41), the level of ADAMTS-13 mRNA in 0. 01 μg/ml LPS stimulation group was markedly decreased at 48 hours (18.78±0.86, P〈0.01). At 24 hours, the levels of ADAMTS-13 mRNA (23.43± 0.63, 22.41±0.76) were markedly decreased in 0.1 μg/ml and 1μg/ml LPS stimulation groups (P〈0.05 and P〈0.01). The level of ADAMTS-13 mRNA (20.01 ± 2.47) in 5 μg/ml LPS stimulation group was markedly decreased at 12 hours (P 〈 0.01). Compared with the control group [(115.76 ± 2.36) ng/ml], protein level of ADAMTS-13 [(113.43± 1.07) ng/ml] was markedly decreased at 12 hours in 0.01 μg/ml LPS stimulation group (P〈 0.05). The protein level of ADAMTS-13 [(7.63±2.64) ng/ml] was lowest in 5 μg/ml LPS stimulation group at 72 hours (P〈0.01). Conclusion Normal RAECs can express ADAMTS-13 at both mRNA and protein to certain extent. The expression of ADAMTS-13 mRNA and protein are decreased after LPS challenge in different concentrations for different duration in dose-dependent and time-dependent manner.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2011年第8期482-485,共4页
Chinese Critical Care Medicine
基金
天津市医药卫生科研基金资助项目(09KZ62)
天津市中西医结合科研项目(2005088)
关键词
脂多糖
主动脉内皮细胞
血管性血友病因子裂解酶
大鼠
Lipopolysaecharide
Aortic endothelial cell
Von Willebrand factor cleaving protease
Rat