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中华妇幼临床医学杂志(电子版) ›› 2016, Vol. 12 ›› Issue (01) : 47 -55. doi: 10.3877/cma.j.issn.1673-5250.2016.01.009

所属专题： 文献；

论著

上皮细胞钠离子通道在子痫前期患者胎盘组织中的表达及其与子痫前期发病的关系
杨悦1, 王姗2, 刘兴会2, 潘天颖2, 何国琳2,*,*()   
  1. 1. 610041　成都，四川大学华西临床医学院
    2. 610041　成都，四川大学华西第二医院妇产科
  • 收稿日期:2015-09-15 修回日期:2016-01-05 出版日期:2016-02-01
  • 通信作者: 何国琳

Diverse expression of epithelial sodium channel on placenta tissue of preeclampsia patients and its relationship with preeclampsia

Yue Yang1, Shan Wang2, Xinghui Liu2, Tianying Pan2, Guolin He2()   

  1. 1. Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
  • Received:2015-09-15 Revised:2016-01-05 Published:2016-02-01
  • Corresponding author: Guolin He
  • About author:
    corresponding author: He Guolin, Email:
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引用本文：

杨悦, 王姗, 刘兴会, 潘天颖, 何国琳. 上皮细胞钠离子通道在子痫前期患者胎盘组织中的表达及其与子痫前期发病的关系[J]. 中华妇幼临床医学杂志(电子版), 2016, 12(01): 47-55.

Yue Yang, Shan Wang, Xinghui Liu, Tianying Pan, Guolin He. Diverse expression of epithelial sodium channel on placenta tissue of preeclampsia patients and its relationship with preeclampsia[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2016, 12(01): 47-55.

目的

上皮细胞钠离子通道(ENaC)在子痫前期患者胎盘中的表达及其与子痫前期(PE)发病的关系。

方法

选择2009年3月至2011年3月在四川大学华西第二医院行剖宫产分娩的92例产妇的胎盘组织为研究对象，根据孕妇是否患有重度子痫前期，将其分别纳入重度PE组(n＝48，重度PE患者)和对照组(n＝44，正常妊娠者)。此外，选择于早孕期(孕龄为8～11孕周)因计划外妊娠等因素要求行人工终止妊娠孕妇的胎盘组织纳入早孕期组(n＝36)。早孕期组的胎盘组织取自选择性负压吸引人工终止妊娠术者。重度PE组和对照组产妇行剖宫产分娩待胎盘娩出后，立即取胎盘母体面的中央绒毛区组织。3组受试者的住院时间等比较，差异无统计学意义(P>0.05)。体外实验在正常人滋养层细胞株HTR-8/Svneo上进行。采用免疫组化技术(SP法)检测ENaC在孕妇胎盘组织的定位。实时荧光定量逆转录聚合酶链反应法(qPCR)及Western blotting法检测胎盘组织ENaC mRNA及蛋白的表达水平。此外，观察雌、孕激素对滋养细胞HTR-8/SVneo ENaC表达的调节作用。再利用小干扰RNA(SiRNA/ENaC)干预ENaC的表达后，通过划痕实验及侵袭实验观察HTR-8/Svneo细胞迁移及侵袭能力的变化。本研究遵循的程序符合四川大学华西第二医院人体试验委员会所制定的伦理学标准，得到该委员会批准，标本的采集过程均征得受试对象同意，并与受试对象签署临床研究知情同意书。

结果

①重度PE组和正常妊娠组孕妇孕前体质指数(BMI)、孕期体重增加、基础收缩压、晚孕期收缩压、孕期收缩压增加、基础舒张压、晚孕期舒张压、孕期舒张压增加、新生儿出生体重等一般临床资料比较，差异均有统计学意义(t＝2.586、2.16、2.139、15.817、13.078、4.896、16.600、11.119、8.456，P<0.05)。②免疫组化法结果显示，ENaCα亚基主要表达于早孕期胎盘合体滋养细胞及绒毛滋养细胞的细胞膜上。③ qPCR结果表示：ENaC各亚基在不同妊娠时期的胎盘组织中均有表达，但其表达水平存在差异，其中ENaC α亚基主要在早孕期表达，而ENaC β亚基主要在对照组表达。ENaC β亚基在重度PE组的相对表达水平为0.48±0.17，显著低于ENaC β亚基在正常妊娠组中的相对表达水平为0.89±0.20，并且差异有统计学意义(t＝2.465，P<0.05)。④雌激素(1 μmol/L)、孕激素(5 μmol/L)可下调ENaC mRNA在滋养细胞HTR-8/SVneo上的表达水平，孕激素(5 μmol/L)可下调ENaC在蛋白水平的表达水平。⑤ ENaC蛋白水平的表达被ENaC α亚基特异性小干扰RNA(siRNA/ENaC)下调后，HTR-8/SVneo细胞的迁移及侵袭能力减弱。

结论

晚孕期胎盘组织中以ENaCβ亚基表达为主，重度PE患者ENaC β亚基表达水平较正常妊娠者降低；下调ENaC的表达滋养层细胞侵袭与细胞迁移的能力减弱，推测ENaC可能通过影响滋养细胞的迁移及侵袭参与PE发病。

关键词: 上皮细胞, 钠通道, 先兆子痫, 胎盘, 细胞迁移分析, 妇女
Objective

To explore the expression of epithelial sodium channel (ENaC) in placenta tissue of preeclampsia (PE) patients, and discuss the relationship between ENaC and PE.

Methods

From March 2009 to March 2011, a total of 92 pregnant women who received cesarean section at West China Second University Hospital, Sichuan University were included into this study. They were divided into two groups according to whether they had PE or not, severe PE group (n= 48) and control group (n=44). Meanwhile, another 36 pregnant women who underwent induced abortion because of social factors were also included into this study as early pregnant group (n=36). In vitro study was conducted on human extrovilloustrophoblast cell lines HTR-8/Svneo cells. Immunohistochemistry streptavidin-perosidase (SP) method was used to study the localization of ENaC of placenta tissues; quantitative real-time polymerase chain reaction (qPCR) and Western blotting were used to study mRNA and protein expression of ENaC in placenta tissues of these patients and extrovilloustrophoblast cell lines HTR-8/SVneo cells which cultured in a condition of estrogen or progesterone. The migration and invasion assay were used to further study the variation of HTR-8/SVneo's migration and invasion ability when cells were treated with ENaC α subunit specific RNA (siRNA). The study protocol was approved by the Ethical Review Board of Investigation in Human Being of West China Second University Hospital, Sichuan University. Informed consents were obtained from each patient.

Results

① There were significant differences between severe PE group and control group in the aspects of body mass index (BMI)before pregnant, the increase of weight during pregnancy, basic systolic pressure, systolic pressure during late pregnancy, the increase of systolic pressure during pregnancy, basic diastolic pressure, diastolic pressure during late pregnancy, the increase of diastolic pressure during pregnancy, and neonate birth weight (t=2.586, 2.16, 2.139, 15.817, 13.078, 4.896, 16.600, 11.119, 8.456; P<0.05). ② Immunohistochemistry result showed that ENaC was selectively expressed in placental trophoblast cells in the plasma membrane; ENaC α subunit was mainly expressed on first term placenta, while ENaC β subunit mainly expressed on third term placenta. The relative expression level of ENaC β subunit in severe PE group and control group were 0.48±0.17 and 0.89±0.20, which had significant difference (t=2.465, P<0.05). ④ Compared with normal pregnancy, the protein levels of ENaC was significantly reduced in the patients in severe PE group. Estrogen (1 μmol/L) could down-regulate the expression of ENaC α subunit mRNA on HTR-8/SVneo cells, while progesterone (5 μmol/L) could down-regulate the expression of ENaC α subunit on both mRNA and protein levels. ⑤ Cell migration and invasion ability was weakened when ENaC was down-regulated by siRNA.

Conclusions

The epithelial sodium channel may be involved in the pathogenesis of PE through regulating the migration and invasion ability of trophoblast cells.

Key words: Epithelial cells, Sodium channels, Pre-eclampsia, Placenta, Cell migration assays, Women
图1 ENaC α亚基在早孕期组和对照组胎盘组织中的表达(图1A: ENaC α　亚基表达于早孕期组胎盘合体滋养细胞及绒毛滋养细胞的细胞膜上；图1B：ENaC α　亚基在对照组胎盘合体滋养细胞上的表达较弱；图1C：早孕期胎盘的IgG对照；图1D：对照组胎盘的IgG对照)(SP法，高倍镜，标尺＝40 μm)
图2 ENaC各亚基在胎盘组织上的表达(图2A：ENaC　各亚基在早孕期胎盘组织中的表达；图2B：ENaC　各亚基在对照组胎盘组织中的表达)
图3 Western blotting法检测ENaC β亚基蛋白电泳图
图4 两组孕妇胎盘组织中ENaC β亚基蛋白相对表达水平
表1 重度PE组和对照组孕妇一般临床资料比较(±s)
组别 例数 孕前BMI(kg/m2) 孕期体重增加(kg) 分娩前BMI(kg/m2) 基础收缩压(mmHg) 晚孕期收缩压(mmHg)
重度PE组 48 21.9±2.7 15.1±3.6 27.9±3.4 113.5±7.9 161.5±21.3
对照组 36 20.6±2.9 16.6±3.9 27.1±2.9 110.1±9.1 111.9±9.7
t值 　 2.586 2.160 1.389 2.139 15.817
P值 　 0.011 0.033 0.168 0.035 <0.001
组别 孕期收缩压增加(mmHg) 基础舒张压(mmHg) 晚孕期舒张压(mmHg) 孕期舒张压增加(mmHg) 新生儿出生体重(g)
重度PE组 49.2±23.9 74.4±1.9 102.9±14.4 28.9±16.7 2 381±662
对照组 9.4±2.1 70.1±6.4 70.4±8.4 7.4±2.5 3 253±393
t值 13.078 4.896 16.600 11.119 8.456
P值 <0.001 <0.001 <0.001 <0.001 <0.001
图5 qPCR结果显示雌、孕激素降低滋养细胞上ENaC α亚基mRNA水平的表达
图6 Western blotting法显示孕激素显著降低滋养细胞上ENaC α亚基蛋白水平的表达
图7 不同转染浓度下HTR-8/SVneo细胞系中ENaC α亚基蛋白电泳图
图8 siRNA转染细胞后HTR-8/SVneo细胞迁移能力的变化(图8A、B：实验组迁移细胞数明显减少；图8C、D：阴性对照组迁移细胞数量)
图9 实验组(siRNA/ENaC)和对照组(siRNA/Ctrl)的迁移细胞数
图10 siRNA转染细胞后HTR-8/SVneo细胞侵袭能力的变化(图10A：实验组，低倍镜；图10B：阴性对照组，低倍镜；图10C：空白对照组，低倍镜；图10D：实验组，高倍镜；图10E：阴性对照组，高倍镜；图10F：空白对照组，高倍镜)(SP法)
图11 各组穿过人工基底膜的HRT-8/SVneo细胞数
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