CAJ | 학술논문

目的从剂量-效应和时间-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、5 μg/ml LPS刺激组,分别于12、24、48、72 h用半定量逆转录-聚合酶链反应(RT-PCR)测定内皮细胞ADAMTS-13 mRNA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-13 mRNA和蛋白表达逐渐下降.与空白对照组(25.22±1.41)比较,0.01μg/ml LPS刺激48 h时ADAMTS-13 mRNA表达(18.78±0.86)即明显下降(P＜0.01);0.1μg/ml和1μg/ml LPS刺激24 h时ADAMTS-13 mRNA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12 h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/ml]比较,0.01 μg/ml LPS刺激12 h时ADAMTS-13蛋白表达[(113.43±1.07)ng/ml]即明显下降(P＜0.05);至5 μg/ml LPS刺激72 h时ADAMTS-13蛋白表达[(7.63±2.64)ng/ml]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-13 mRNA和蛋白表达降低,具有剂量依赖性和时间依赖性.
목적 종제량-효응화시간-효응관계탐토지다당(LPS)대대서주동맥내피세포(RAECs)혈관성혈우병인자렬해매(ADAMTS-13)mRNA화단백표체적영향.방법 채용조직첩괴법배양Wistar대서적RAECs,1주후안1∶3전대지제4～5대,장세포분위공백대조조화0.01、0.1、1、5 μg/ml LPS자격조,분별우12、24、48、72 h용반정량역전록-취합매련반응(RT-PCR)측정내피세포ADAMTS-13 mRNA표체;용매련면역흡부법(ELISA)검측상청액내ADAMTS-13단백수평.결과 공백대조조유일정량적ADAMTS-13mRNA화단백표체.수착LPS자격농도증가화자격시간연장,ADAMTS-13 mRNA화단백표체축점하강.여공백대조조(25.22±1.41)비교,0.01μg/ml LPS자격48 h시ADAMTS-13 mRNA표체(18.78±0.86)즉명현하강(P＜0.01);0.1μg/ml화1μg/ml LPS자격24 h시ADAMTS-13 mRNA표체(23.43±0.63、22.41±0.76)즉명현하강(P＜0.05화P＜0.01);5μg/ml LPS자격12 h시ADAMTS-13 mRNA표체(20.01±2.47)즉명현하강(P＜0.01).여공백대조조[(115.76±2.36)ng/ml]비교,0.01 μg/ml LPS자격12 h시ADAMTS-13단백표체[(113.43±1.07)ng/ml]즉명현하강(P＜0.05);지5 μg/ml LPS자격72 h시ADAMTS-13단백표체[(7.63±2.64)ng/ml]강지최저(P＜0.01).결론 RAECs유일정량적ADAMTS-13mRNA화단백표체;불동농도LPS자격내피세포불동시간후ADAMTS-13 mRNA화단백표체강저,구유제량의뢰성화시간의뢰성.
Objective To investigate the dose-response effect and time-response effect of lipopolysaccharide (LPS) on von 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.were randomly divided into five groups.: control group and four LPS stimulation groups (0. 01, 0. 1, 1and 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 1 μg/ml LPS stimulation groups (P＜0.05 and P＜0.01). The level of ADAMTS-13 mRNA (20.01± 2.47) in 5μg/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 5μg/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.