活化蛋白C对脂多糖诱导大鼠主动脉内皮细胞表达血管性血友病因子及其裂解酶的影响

The effects of activated protein C on the von Willebrand factor and von Willebrand factor cleaving protease of rat aortic endothelial cell induced by lipopolysaccharide

目的探讨活化蛋白C（APC）对脂多糖（LPS）诱导大鼠主动脉内皮细胞（RAECs）血管性血友病因子抗原（vWFAg）及其裂解酶（ADAMTS-13）蛋白表达的影响。方法采用组织贴块法培养Wist~大鼠的RAECs，1周后传代至第4．5代用于实验。将细胞分为对照组、LPS刺激组（1mg／L）以及APC干预组（在LPS刺激后分别加入终浓度为0．1、1、10mg／L的APC）；分别于12、24、48、72h采用酶联免疫吸附试验（ELISA）测定RAECs培养上清液中vWFAg和ADAMTS-13蛋白的表达水平。结果对照组仅有少量的vWFAg和ADAMTS-13蛋白表达。随LPS刺激时间延长，vWFAg表达12h即明显增多，48h达峰值[（285．45±30．13）％]，ADAMTS-13蛋白表达（μg／L）呈降低趋势，72h降至最低（13．32±2．37），与对照组[vWFAg：（94．53±7．83）％，ADAMTS-13：115．76±2．36]比较差异有统计学意义（均P〈0．01）。APC可逆转LPS刺激后对vwF她的促进作用和对ADAMTS-13的抑制作用，且剂量越大，逆转效果越明显。10mg／LAPC干预后可使LPS刺激48h时达峰值的vWFAg明显降低[（198．43±17．92）％比（285．45±30．13）％]，使LPS刺激72h时降至最低的ADAMTS-13（μg／L）明显升高（125．25±2．70比13．32±2．37），差异有统计学意义（均P〈0．01）。结论LPS刺激RAECs后vWFAg明显升高，ADAMTS-13蛋白表达明显降低，具有时间依赖性；用不同浓度APC干预后，随浓度增加和作用时间延长，vWFAg明显降低，ADAMTS-13蛋白表达明显升高，具有剂量依赖性和时间依赖性。

Objective To investigate the activated protein C （APC） on the yon Willebrand factor antigen （vWFAg） and yon Willebrand factor cleaving protease （ADAMTS-13） protein expression in rat aortic endothelial cells （RAECs） induced by lipopolysaccharide （LPS）. Methods RAECs from Wistar rats were cultured with the tissue explants adherence method. RAECs were cultured for one week, After one week culture, RAECs in 4-5 generations were divided into control group, LPS stimulation groups （ 1 mg/L） and APC intervention groups （0.1, 1 and 10 mg/L APC was added after LPS stimulation）. The supernatants were obtained at 12, 24, 48, and 72 hours after LPS stimulated to determine the vWFAg and protein of ADAMTS-13 expression by enzyme-linked immunoadsorbent assay （ELISA）. Results In the control group, RAECs expressed little vWFAg and protein of ADAMTS-13. With stimulation of LPS, the vWFAg was significantly increased at 12 hours, and reached the peak at 48 hours [ （285.45 ± 30.13）% ], and the level of ADAMTS-13 （μg/L） was gradually decreased, and reached the nadir at 72 hours （13.32 ± 2.37）, there was significant difference compared with control group [vWFAg： （94.53 ± 7.83）%, ADAMTS-13：115.76 ± 2.36, both P〈0.01 ）. The effects on vWFAg promoting and ADAMTS-13 inhibition after LPS stimulation could be dose-dependendy reversed by APC. 10 mg/L of APC could decrease the peak of vWFAg at 48 hours of LPS stimulation [ （198.43 ± 17.92）% vs. （285.45 ± 30.13 ）% ], and increase the minimize of ADAMTS-13 （txg/L） at 72 hours of LPS stimulation （125.25 ± 2.70 vs. 13.32 ± 2.37）, with significant difference （both P〈0.01 ）. Conclusions After stimulation with LPS, the level of vWFAg was time-dependent increased, as the protein of ADAMTS-13 was decreased. APC could attenuate the effect of LPS on vWFAg and protein of ADAMTS-13 with dose-dependent and time-dependent patterns.