动脉粥样硬化发生过程中C反应蛋白对内皮细胞功能的影响:量效与时效趋势

Dose-dependent and time-dependent effects of C reactive protein on the endothelial function during the occurrence of atherosclerosis

目的:探讨C反应蛋白对内皮细胞株304细胞释放前列环素、纤溶酶原激活物抑制剂1的影响。方法:实验于2005-01/2005-04在解放军第305医院老年病中心实验室完成。将内皮细胞株304细胞(第131代)以3×104细胞悬液接种到96孔板中,细胞融合生长后换用含0mg/L(对照组),25和50及100mg/L的C反应蛋白Dulbecco改良的Eagle培养基,作用24h,观察C反应蛋白的量效。加入50mg/LC反应蛋白,作用3,6,12,24h,观察C反应蛋白时间效应。用酶联免疫吸附法检测培养液上清液中前列环素的稳定代谢产物6-酮-前列环素F1α、纤溶酶原激活物抑制剂1的水平。结果:①量效关系:C反应蛋白各质量浓度组纤溶酶原激活物抑制剂1水平明显高于对照组(P<0.01),并随C反应蛋白质量浓度增加而增加,尤以100mg/L浓度较25mg/L时增加明显(P<0.05);C反应蛋白各质量浓度组6-酮-前列腺素F1α水平明显低于对照组(P<0.01),并随C反应蛋白质量浓度增加而下降(P<0.01)。②时效关系:C反应蛋白(50mg/L)作用内皮细胞后,随时间延长纤溶酶原激活物抑制剂1水平呈递增趋势(P<0.05～0.01)。作用12h内6-酮-前列腺素F1α水平随时间延长而呈递增趋势(P<0.01),24h明显小于12h(P<0.05);3h时大于对照组(P<0.05),12和24h时明显少于对照组(P<0.01)。结论:C反应蛋白可能通过抑制前列腺环素生成,促进纤溶酶原激活物抑制剂1释放参与急性冠脉综合征的病理生理过程。损伤刺激内皮细胞早期能增加前列腺环素的释放,以提高对损伤的防御,随着时间的延长,防御机制逐渐耗竭,内皮细胞合成前列腺环素被抑制,释放前列腺环素减少。

AIM:To investigate the effect of C reactive protein on prostaglandin and plasminogen activator inhibitor-1 in endothelial cell strain 304 cells.METHODS:The experiment was carried out in the Military Central Laboratory of Geriatrics,the 305 Hospital of Chinese PLA from January to April 2004.Endothelial cell strain 304 cells(the 131st generation) were inoculated to the 96-cell plate with 3× 104 cell suspension;After growth of cell fusion,the Dulbecco modified Eagle medium containing 0 mg/L(control group),25,50,100 mg/L C reactive protein for 24 hours,and the dose dependent effects of C reactive protein were observed.50 mL C reactive protein was added for 3,6,12 and 24 hours to observe its time dependent effects.The levels of the steady metabolite 6 keto prostaglandin F1 alpha and plasminogen activator inhibitor 1 in the supernatant of culture medium were detected with enzyme linked immunoabsorbent assay(ELISA).RESULTS:① Dose dependent association: The level of plasminogen activator inhibitor 1 was obviously higher in the each mass concentration C reactive protein group than in the control group(P< 0.01),and it was increased with the increase of the mass concentration of C reactive protein, and it was more obvious when the concentration was 100 and 15 mg/L(P< 0.05);The level of 6 keto prostaglandin F1 alpha was obviously lower in each mass concentration C reactive protein group than in the control group(P< 0.01),and it was decreased with the increase of the mass concentration of C reactive protein(P< 0.01).② Time dependent association:After the treatment of C reactive protein(50 mg/L),the level of plasminogen activator inhibitor 1 in endothelial cells was gradually increased with he prolongation of time(P< 0.05 to 0.01);Within 12 hours,the level of 6 keto prostaglandin F1 alpha was gradually increased with the prolongation of time(P< 0.01),it was obviously lower at 24 hours than at 12 hours(P< 0.05);At 3 hours,it was higher than that in the control group(P< 0.05);At 12 and 24 hours,it was markedly lower than that in the control group(P< 0.01).CONCLUSION:C reactive protein may be involved in the pathophysiological process of acute coronary syndrome by inhibiting the generation of prostacyclin and promoting the release of plasminogen activator inhibitor 1.Injury and stimulation of endothelial cells at early stage can increase the release of prostacyclin, and then improve the defense to the injury;With the prolongation of time,the defense mechanism was gradually exhausted,the endothelial cells synthetized prostacyclin is inhibited,and its release was reduced.