细胞外信号调节蛋白激酶通路在胎盘生长因子1诱导脐静脉内皮细胞释放一氧化氮中的作用

Effect of extracellniar signal-regulated kinase pathway on nitric oxide release by human umbilical vein endothelial cell induced by placental growth factor-1

目的探讨细胞外信号调节蛋白激酶（ERK）通路在胎盘生长因子1（PLGF-1）诱导脐静脉内皮细胞（HUVEC）释放一氧化氮（NO）中的作用。方法选择因头盆不称、胎儿窘迫或胎位异常行剖宫产分娩的新生儿50例，采用胰蛋白酶消化法进行脐带HUVEC原代培养。（1）培养成功后，用免疫组化法通过Ⅷ因子鉴定细胞形态；（2）用蛋白印迹法和RT—PCR技术检测HUVEC中酪氨酸激酶受体1（Flt-1）蛋白和mRNA表达；（3）用浓度为10ng／ml的PLGF-1培养细胞（观察A组），并在培养前及培养后2．5、5、10、20min收集细胞，提取蛋白，用蛋白印迹法检测ERK蛋白的表达；（4）用浓度为10ng／ml的PLGF-1培养细胞，收集培养后20、40、160、360、480、720、1440min的细胞培养液，用硝酸盐还原酶法检测培养液中NO的含量；（5）先用含ERK特异性抑制剂——PD98059（100μmol／L）的培养基培养细胞60min后，换含PLGF-1的培养基继续培养（观察B组），收集培养后20、40、160、360、480、720、1440min的细胞培养液，用硝酸盐还原酶法检测培养液中NO的含量。以无血清培养基培养的细胞为对照组，细胞处理时间、培养条件和收集细胞液时间同观察组。实验重复3次。结果（1）免疫组化鉴定培养的细胞为HUVEC。（2）HUVEC中有Fit-1mRNA和蛋白的表达。（3）观察A组PLGF-1培养HUVEC后2．5min即有ERK蛋白表达强度的升高，5min时表达强度达到高峰，10min时表达强度降低。（4）PLGF-1培养20min后HUVEC培养液中NO含量开始升高，为（6．96±0．34）μmol／L，培养40min时NO含量为（9．45±0．59）μmol／L，培养360min时NO达（15．82±0．69）μmol／L，各时间点NO含量比较，差异均有统计学意义（P〈0．05）。（5）观察B组NO释放显著受到抑制，从培养160min到1440min，NO含量下降达50％以上。结论ERK通路可能是PLGF诱导HUVEC释放NO的重要信号通路之一。

Objective To investigate the effect of extracellular signal-regulated kinase （ERK） pathway on nitric oxide （NO） release by human umbilical vein endothelial cell （HUVEC） induced by placental growth factor-1 （PLGF-1）. Methods During January to April 2006, 50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position. HUVEC were primarily cultured by trypsin digestion. Then the following procedures were performed： （1） Cells were identified using the morphology and Ⅷ factor immunohistochemistry methods if the culture was satisfactory. （2） Cells were collected, and fms-like tyrosin kinase （ Flt-1 ） protein and its mRNA expression were detected with immunoprints and RT-PCR methods. （3） The protein was extracted after cells were treated with PLGF-1 （cells were collected before the treatment and 2. 5, 5,10, 20 min after the treatment）. The protein levels of ERK were determined by immunoprints. （4） The cells were cultured with serum-free culture medium containing PLGF-1 only （culture media were collected 20, 40,160, 360,480, 720 and 1440 min after the treatment） . The quantity of NO was detected with nitrate reductase method. （5） The cells were cultured with serum-free culture medium containing PD98059, the inhibitor of mitogen-activated protein kinase （MAPK）/MEK for 60 min. Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 min. The culture media were collected. The quantity of NO was detected by nitrate reductase method. The samples were divided into treatment group and control group. Control group was exactly the same in treatment time, culture condition, and time to collect the cells as the treatment group, except that it was not treated with PLGF-1 or PD 98059. Results （ 1 ） By morphology and Ⅷ factor immunohistochemistry the cultured cells were identified to be HUVEC. （2） Flt-1 mRNA and protein were expressed in HUVEC. （3） Expression of ERK protein started to increase at 2. 5 min after treatment of HUVEC with PLGF-1, reaching the peak at 5 min, and decreased at 10 min. （4） In comparison with the control group, NO started to increase at 20 min after treatment of HUVEC with PIGF-1 （6. 96 ±0. 34） μmol/L, significantly increased at 40 min （9.45 ±0.59）μmol/L, and arrived at the peak at 480 min （15.82 ±0. 69）μmol/L. Comparison between the two groups showed a significant difference （ P 〈 0. 05 ）. （5） Release of NO from the cells treated with PD98059 for 1 hour and PLGF was significantly inhibited, compared with the cells treated with PLGF-1 only. Conclusion ERK pathways play an important role in NO release by HUVEC induced by PLGF.