ACK1在子痫前期孕妇胎盘中的表达及其对滋养细胞侵袭功能的调控

Expression of activated Cdc42-associated kinase in the placenta of pregnant women with preeclampsia and its regulatory effect on invasion of trophoblasts

摘要:

目的：研究活化的Cdc42结合激酶（activated Cdc42 associated kinase，ACK1）在正常足月孕妇胎盘与子痫前期（preeclampsia，PE）孕妇胎盘组织中的表达差异及其对滋养细胞功能的调控作用，探讨其在子痫前期发病机制中的作用。方法：收集2015年10月至2016年10月在重庆医科大学附属第一院产科行计划性剖宫产手术的23例正常足月孕妇胎盘和23例PE孕妇胎盘组织，使用免疫组化（immunohistochemistry，IHC）及蛋白免疫印迹法（Western blot，WB）检测胎盘组织中ACK1的表达及差异情况。滋养细胞株（HTR8/SVneo）分为3组：常氧对照组（N组）、缺氧/复氧组（H/R组）及ACK1 基因序列慢病毒敲降组（ACK1 shRNA组），使用Transwell实验分析检测各组细胞的侵袭情况，采用流式细胞仪检测各处理组细胞凋亡率。使用West－ern blot检测各组细胞中ACK1蛋白，金属蛋白质酶（matrix metalloproteinase，MMP）-9及其特异性抑制因子（the tissue inhibitor of metalloproteinase，TIMP）-2的蛋白表达差异。结果：与正常足月孕妇胎盘比较，ACK1在人类子痫前期胎盘中表达明显降低（t=3.890，P=0.018）。细胞学实验中，与N组比较，ACK1 shRNA组和H/R组ACK1蛋白表达明显降低（t=12.260，P=0.000；t=10.740，P=0.000），ACK1 shRNA组和H/R组MMP9蛋白表达均明显降低（t=8.071，P=0.000；t=7.745，P=0.001），TIMP1蛋白表达显著升高（t=10.690，P=0.000；t=14.330，P=0.000）。ACK1 shRNA组和H/R组细胞迁移至下室的细胞数明显减少（t=12.260，P=0.000；t=11.320，P=0.000），H/R组细胞凋亡率显著升高（t=2.260，P=0.000），但ACK1 shRNA组细凋亡率无明显改变（t=8.317，P=0.088）。结论：ACK1的表达下调可抑制滋养细胞的侵袭，可能在子痫前期发病机制中有重要的作用。

Abstract:

Objective：To investigate the expression of activated Cdc42-associated kinase（ACK1） in the placenta of normal women with full-term pregnancy versus pregnant women with preeclampsia（PE），the regulatory effect of ACK1 on invasion of trophoblasts，and the role of ACK1 in the pathogenesis of PE. Methods：Placenta tissue samples were collected from 23 normal women with full-term pregnancy and 23 pregnant women with PE who underwent elective cesarean section in The First Affiliated Hospital of Chongqing Medical University from October 2015 to October 2016. Immunohistochemistry and Western blot were used to measure the expression of ACK1 in the placenta. The trophoblast cell line（HTR8/SVneo cells） was divided into normoxia control group（N group），hypoxia/reoxygenation（H/R） group，and ACK1 lentivirus knockdown group（ACK1 shRNA group）. Transwell assay was used to evaluate the invasion of cells and flow cytometry was used to measure the apoptosis of cells. Western blot was used to measure the protein expression of ACK1，matrix metalloproteinase-9（MMP-9），and tissue inhibitor of metalloproteinase-2（TIMP2） in HTR8/SVneo cells. Results：The expression of ACK1 in the placenta of PE patients was significantly lower than that in the placenta of normal women with full-term pregnancy（t=3.890，P=0.018）. Compared with the N group，the ACK1 shRNA group and the H/R group had significant reductions in the protein expression of ACK1（t=12.260，P=0.000；t=10.740，P=0.000） and MMP-9（t=8.071，P=0.000；t=7.745，P=0.001） and a significant increase in the protein expression of TIMP1（t=10.690，P=0.000；t=14.330，P=0.000）. The ACK1 shRNA group and the H/R group had a significant reduction in the number of cells migrating to the lower chamber（t=12.260，P=0.000；t=11.320，P=0.000）. The H/R group had a significant increase in cell apoptosis rate（t=2.260，P=0.000），while the ACK1 shRNA group had no significant change in apoptosis rate（t=8.317，P=0.088）. Conclusion：Downregulation of ACK1 expression inhibits the invasion of trophoblasts，suggesting that ACK1 may play an important role in the pathogenesis of PE.