重组尿激酶原对人肺动脉内皮细胞u-PA系统的影响

Effects of Recombinant Pro-urokinase on Expression of Urokinase Type Plasminogen Activator System in Human Pulmonary Arterial Endothelial Cells

目的 探讨重组尿激酶原对体外培养的正常人肺动脉内皮细胞(HPAECs)尿激酶型纤溶酶原激活物系统表达的影响.方法 将重组尿激酶原0或150 IU/mL与人肺动脉内皮细胞共同孵育8 h,收集培养上清并应用ELISA方法检测其中尿激酶型纤溶酶原激活物受体(u-PAR)和纤溶酶原激活物抑制物-1(PAI-1)的含量;将重组尿激酶原(0～150 IU/mL)分别与HPAECs共同孵育(0～24 h),提取细胞总RNA并应用RT-PCR技术检测尿激酶型纤溶酶原激活物(u-PA)mRNA表达的变化.结果 细胞培养上清ELISA实验表明,与对照组相比,重组尿激酶原150 IU/mL组细胞培养液中u-PAR的含量显著增加[(0.51±0.04)μg/L vs (0.58±0.05) μg/L,P=0.005];PAI-1的含量显著下降[(66.75±7.92) μg/L vs (53.38±12.18) μg/L,P=0.009].RT-PCR结果表明,HPAECs与重组尿激酶原150 IU/mL共同培养后0 h、4 h、8 h、12 h和24 h 5个时间点u-PA条带与GAPDH条带平均光密度之比分别为(0.34±0.11)、(0.51±0.12)、(0.58±0.12)、(0.50±0.18)和(0.35±0.10).其中8 h组与对照组相比差异有统计学意义,8 h组与24 h相比P=0.053.结论 重组尿激酶原能够明显促进HPAECs释放u-PAR并显著抑制PAI-1的表达和释放.重组尿激酶原能够呈时间依赖性提高u-PA mRNA在HPAECs的表达.重组尿激酶原直接影响人肺动脉内皮细胞u-PA系统的表达,该作用可能是增强药物本身溶栓疗效的一种重要途径.

Objective Pro-urokinase,a new thrombolytic agent that is currently being evaluated for the treatment of myocardial infarction,stroke and pulmonary embolism.However little research has been done about the effects of recombinant pro-urokinase on endothelial cells.The purpose of this article is to study the effects of pro-urokinase on the expression and release of urokinase type plasminogen activator receptor(u-PAR) and plasminogen activator inhibitor1(PAI-1) and to study the effects of pro-urokinase on the mRNA expression of urokinase type plasminogen activator(u-PA) in human pulmonary arterial endothelial cells(HPAECs).Methods The HPAECs were cultured in M200 medium with low serum growth supplement until became confluent.Then the HPAECs were starvated for 12 h,and ready to be used in different experiments.(1) HPAECs were incubated with pro-urokinase(0 or 150 IU/mL) for 8 h,then the medium was centrifuged for 10 min at 1 000 r/min and expression of u-PAR or PAI-1 in the medium detected with ELISA kits.(2) HPAECs was incubated with pro-urokinase 150 IU/mL for 0,4,8,12 and 24 hours.In each group,the total RNA of HPAECs was extracted by Trizol reagent and the gene expression of u-PA mRNA detected with RT-PCR and compared with that of GAPDH mRNA.All cells in these experiments were of 3 to 6 passages.Results The cells were flat with round or oval nuclei containing several prominent nucleoli.Six to seven days after plating,the cultured HPAECs formed a monolayer.In the ELISA tests,the u-PAR content in pro-urokinase treated cell was statistically increased as compared with the control ones((0.51±0.04)μg/L vs(0.58±0.05)μg/L,P=0.005);the PAI-1 content in serum was statistically decreased in pro-urokinase treated cells as compared with control ones((66.75±7.92)μg/L vs(53.38±12.18)μg/L,P=0.009).In RT-PCR experiments,after incubation with pro-urokinase 150 IU/mL for increasing hours(0,4,8,12 and 24 hours),the u-PA bd/GAPDH band ratio was(0.34±0.11),(0.51±0.12),(0.58±0.12),(0.50±0.18) and(0.35±0.10) respectively.Pro-urokinase(150 IU/mL) incubation could up-regulate the expression of u-PA mRNA in HPAECs in a time-dependent manner and the highest expression was shown at 8 hours.Conclusion Under normal conditions,HPAECs cue could express or release u-PA,u-PAR,and PAI-1.Pro-urokinase could inhibit the release of PAI-1 and enhance the release of u-PAR from cell surface.Pro-urokinase could enhance the expression of u-PA mRNA in HPAECs in a time-dependent manner.Pro-urokinase has direct effects on the expression of u-PA system in HPAECs,which may thus enhance thrombolytic process.