高静水压培养对人脐静脉内皮细胞t-PA和PAI-1的影响

目的探讨高静水压培养对人脐静脉内皮细胞（HUVECs）t—PA和PAI-1的影响及其可能的作用机制。方法选用第4～6代HUVECs，接种于24孔培养板中。依培养压力分为3组：大气压组（0mmHg），中压组（90mmHg）和高压组（180mmHg），每组18份标本。在同一压力组，根据不同药物干预又分为3个亚组：对照组（Ctrl组），双丁酰环磷腺苷组（DBA组，10μmoL／L）和硝普钠组（NP组，10^-4moL／L），每组6份标本。采用ELISA法测定上清液t-PA和PAI-1的抗原浓度，并用细胞内总蛋白进行标化，同时测定细胞内Ca^2＋浓度。结果与大气压组的对照组比较，中压和高压组t-PA浓度均显著降低（P〈0.01），PAI-1浓度显著增高（P〈0.01），t-PA／PAI-1比值显著降低（P〈0.01），Ca^2＋]i也显著增高（P〈0.01）。cGMP诱导剂NP显著降低三个压力组的PAI-1浓度（P〈0.01）和增高t—PA／PAI-1比值（P〈0.05），cAMP的同功异构体DBA显著升高三个压力组的PAI-1浓度（P〈0．01，P〈0．05）。DBA和NP对t-PA浓度没有显著影响（P〉0.05）。结论高静水压可损害内皮细胞的抗纤溶功能。第二信使cAMP、cGMP和细胞内钙离子在此可能起调节作用。

Effects of High Hydrostatic Pressure on the t-PA and PAI-1 Secretions from Vascular Endothelial Cells in Vitro

Objective To investigate the effects of high hydrostatic pressure on the concentrations of t-PA and PAI-1 antigen in the medium culturing human umbilical vein endothelial cells(HUVECs).Methods HUVECs harvested between passages 4 and 6 were used for the experiments.According to the cultured pressure,they were divided into three groups: atmospheric pressure group(0 mmHg),middle pressure group(90 mmHg),and high pressure group(180 mmHg)(18 samples/subgroups).The above three groups each was further divided into three subgroups(6 samples/subgroups),as control group,Na-nitroprusside group(10~(-4) moL/L),DB-cAMP group(10~(-4) moL/L).At the end of the culture period,supernatants were collected to measure the concentrations of t-PA and PAI-1 antigens by ELISA kit.Concentrations of t-PA and PAI-1 antigens were normalized with intracellular proteins.Meanwhile,the concentration of intracellular free calcium(Ca~(2+)]i) was detected.Results Compared with atmospheric pressure group,high hydrostatic pressure induced a dramatic reduction in t-PA antigen and a dramatic increase in PAI-1 antigen(P<0.01).Meanwhile,high hydrostatic pressure significantly increased Ca~(2+)]i(P<0.01).Na-nitroprusside,with a documented cGMP-inducing effect,markedly decreased the concentration of PAI-1 in three groups(P<0.01). The cAMP analogue DB-cAMP markedly increased the concentration of PAI-1 in three groups(P<0.01,P<0.05).Both DB-cAMP and Na-nitroprusside showed no effect on the pressure-induced t-PA reduction(P>0.05).Conclusion High hydrostatic pressure could decrease the level of t-PA antigen and increase the level of PAI-1 antigen in HUVECs culture medium,which results in abnormality of fibrinolytic function.Intracellular calcium,cAMP and cGMP probably act as the mediators for effects of high hydrostatic pressure on t-PA and PAI-1 secretions of endothelial cells.