子痫前期产妇胎盘组织中的氧化应激对Wiskott—Aldrich综合征关联蛋白2表达的影响

Effect of oxidative stress on human Wiskott-Aldrich syndrome related protein 2 expression in placental trophoblasts in preeclampsia

目的探讨子痫前期产妇胎盘组织存在的氧化应激对胎盘滋养细胞Wiskott-Aldrich综合征关联蛋白2（Wiskott-Aldrich syndrome related protein2，WAVE2）表达的影响。方法研究对象为2011年8月15日至2012年2月23日在重庆医科大学附属第一医院分娩的子痫前期产妇20例及正常足月产妇23例（对照组）。剖宫产术后取胎盘组织，采用免疫组织化学法检测胎盘组织WAVE2的定位及分布。采用蛋白质印迹法检测胎盘组织WAVE2的表达，实时荧光定量聚合酶链反应法检测胎盘中WAVE2mRNA的表达，采用组织匀浆法检测2组产妇胎盘组织活性氧（reactive oxygen species，ROs）水平，并对胎盘组织中ROS水平与wAVE2表达水平进行相关性分析。（2）通过构建体外滋养细胞缺氧复氧损伤模型，模拟缺血再灌注诱导的氧化应激损伤。人妊娠早期绒毛外滋养细胞株HTR-8／SVneo细胞贴壁完全后分别置于常氧（常氧组）和缺氧复氧条件（缺氧复氧组）下48h，采用流式细胞仪分析细胞内ROS水平。采用Transwell实验观察细胞迁移侵袭状况。采用细胞免疫荧光法和蛋白质印迹法检测HTR-8／SVneo细胞内WAVE2的定位及表达水平的变化。采用独立样本t检验及Pearson相关检验对数据进行分析。结果（1）子痫前期组24h尿蛋白、母体收缩压及舒张压均高于对照组，新生儿出生体重及胎盘重量均低于对照组[24h尿蛋白：（1．96±0．24）g与（0．08士0．05）g，t=19．436；母体收缩压：（154±13）mmHg与（98±11）FilmHg，t=11．324；母体舒张压：（105±14）mmHg与（69±8）mmHg，t=9．612；新生儿出生体重：（2446±187）g与（3207±233）g，t=16．307；胎盘重量：（432±53）g与（536±67）g，t=14．562；均P〈O．05]。子痫前期组WAVE2mRNA表达低于对照组E（0．28±0．07）与（1．01±0．02），t=12．747]，WAVE2相对表达量低于对照组[（0．63±0．08）与（1．34±0．19），t=11．648]，ROS水平高于对照组[（144．22±12．32）nmol／（mg·prot）与（75．17±8．71）nmol／（mg·prot），t=20．467]，差异均有统计学意义（均P〈0．05）。子痫前期组胎盘组织中ROS水平与wAVE2表达水平呈显著负相关（r=-0．726，P=0．000）。（2）在常氧组中，HTR-8／svneo细胞内的ROS水平为（82．9±5．8）％，而缺氧复氧组为（155．6±8．1）％，高于常氧组（t=12．747，P〈0．05）。缺氧复氧48h后的HTR=8／SVneo细胞与常氧组相比，迁移和侵袭力明显降低[缺氧复氧组分别为（58．4±4．2）％和（51．9±3．3）％，常氧组为100％，t值分别为11．034和13．839，P均〈O．os]。细胞免疫荧光检测结果显示，WAVE2定位于滋养细胞的细胞质中。缺氧复氧48h后，HTR=8／SVneo细胞中WAVE2的表达强度明显弱于常氧组（O．37±0．05与0．76±0．06，t=8．631，P〈0．05）。结论子痫前期胎盘组织中存在过度氧化应激，与胎盘组织中WAVE2表达下调密切相关。缺氧复氧致氧化应激损伤可下调滋养细胞WAVE2的表达；WAVE2表达下调可能损伤滋养细胞迁移侵袭能力，从而参与子痫前期的发生发展。

Objective To explore the effect of oxidative stress on human Wiskott-Aldrich syndrome related protein 2 （WAVE2） expression in placental trophoblasts in women with preeclampsia. Methods （1） Twenty women with preeclampsia and twenty-three normal term pregnant women, delivered from August 15, 2011 to February 23, 2012 in the First Affiliated Hospital of Chongqing Medical University, were recruited and divided into preeclampsia group and control group. Placenta samples were collected after cesarean section. The localization and distribution of WAVE2 in placenta was studied by immunohistochemistry. Quantitative real-time polymerase chain reaction and Western blot were employed to assay the WAVE2 mRNA and protein levels. Tissue homogenates was applied to determine the levels of reactive oxygen species （ROS）. The correlation between ROS levels and WAVE2 was also analyzed. （2） An in vitro hypoxia/reoxygenation （H/R） model was utilized to simulate ischemia/reperfusion injury to placental trophoblasts. The HTR 8/ SVneo cells （immortalized human first trimester extravillous trophoblast cells） were pre incubated overnight, after exposure to H/R or normoxic conditions for 48 hours. Flow cytometry was employed to analyze intracellular ROS level. Meanwhile, Transwell assay was utilized to analyze the invasion and migration of HTR-8/SVneo cells. The location and expression of WAVE2 in trophoblasts was evaluated by cell immunofluorescence and Western blot. Statistical differences between the two groups were evaluated by independent t-tests. Pearson＇s correlation coefficient test was used for correlation analysis. Results （1）Compared with the control group, preeclampsia group had significantly higher 24-hour proteinuria [（1.96±0.24） g vs （0.08±0.05） g, t=19.436, P〈0.051, systolic blood pressure [（154±13） mm Hg vs （98±11） mm Hg, t=11.324, P〈0.05] and diastolic blood pressure [（105±14）mm Hgvs （69＋8） mm Hg,t=9.612, P〈0.05]. In addition, the placental weight and birth weight of infants in preeclampsia group were significantly reduced as compared to the control group E（432±53） g vs （536± 67） g, （2446± 187） g vs （3207±233） g, t=14.562 and 16. 307, allP〈0.05）l. The WAVE2 mRNA level （0.28±0.07 vs 1.01±0.02,t=12.747, P〈 0.05） and the WAVE2 protein levels （0.63±0.08 vs 1.34±0.19, t= 11. 648, P〈0.05） were also significantly decreased in preeclampsia groups. The level of ROS in placenta in the preeclampsia group was significantly higher than in control group [（144.22± 12.32） nmol/（mg· prot） vs （75.17 ± 8.71） nmol/（mg· prot）, t=20. 467, P〈0. 051. There was significant negative correlation between ROS level and WAVE2 protein expression in preeclamptic placenta （r = - 0. 726, P = 0. 000） . （2） In vitro study showed that, the levels of ROS in normoxia group and H/R group was （82.9± 5.8） % and （155.6± 8.1）%, （t= 12. 747, P〈0.05）. Compared with normoxia condition, decreased cell invasion and migration were found in HTR-8/SVneo cells in H/R group [（51.9 ± 3.3）] and （58.4± 4. 2）% respectively, t=11.034 and 13.839, P〈0.057. Results from the cell immunofluorescence showed that WAVE2 protein located in the cytoplasm of HTR-8/SVneo cells, and the expression of WAVE2 protein was significant decreased in HTR-8/SVneo cells after exposure to H/R for 48 h （0.37±0.05 vs 0.76±0.06, t=8.631, P〈0.05）. Conclusions Excessive oxidative stress in preeclamptic placentas was correlated with the decreased expression of WAVE2. H/R-induced oxidative stress could decrease WAVE2 expression, which may contribute to impaired trophoblast invasion and migration in preeclampsia.