脂多糖诱导大鼠主动脉内皮细胞表达血管性血友病因子裂解酶的研究

Induction of expression of yon Willebrand factor cleaving protease of rat aortic endothelial cell by lipopolysaccharide challenge

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.