过表达SATB1保护人滋养细胞氧化应激损伤的机制研究

The mechanism of SATB1 over- expression in protecting human trophoblast from oxidative stress injury

目的探讨富集AT序列的特异性结合蛋白1(SATB1)参与人绒毛外滋养细胞株(HTR8/SVneo)氧化应激损伤的机制。方法收集2013年9月至2014年9月在重庆医科大学附属第一医院产科行选择性剖宫产术的子痫前期孕妇(n=22)和正常足月妊娠行择期剖宫产术的孕妇的胎盘组织(n=22)。采用免疫组化和Western blotting(WB)检测正常晚孕期及子痫前期胎盘组织中SATB1的表达。构建SATB1过表达慢病毒载体(LV-SATB1)和阴性对照载体(LV-NC)。将滋养细胞分为正常培养对照组、缺氧/复氧(H/R)培养组、LV-SATB1+H/R培养组、LV-NC+H/R培养组。利用免疫荧光和WB法检测SATB1在各组细胞中的表达。采用流式细胞仪检测细胞凋亡指数及细胞内活性氧(reactive oxygen species,ROS)水平。利用Transwell小室模型检测细胞的体外侵袭率。结果 SATB1在子痫前期中的表达显著低于正常晚孕组(0.24±0.02 vs.0.12±0.01,t=9.35,P<0.05)。转染LV-SATB1可显著提升H/R培养组的SATB1蛋白水平(0.92±0.17 vs.0.58±0.15,P<0.01)。LV-SATB1可抑制H/R导致的细胞凋亡率上升及ROS聚集(P<0.01)。H/R组细胞侵袭能力低于正常培养组;上调SATB1的表达可提升H/R组细胞的体外侵袭能力(34.33±10.08 vs.19.33±6.52,P<0.05)。结论过表达SATB1可以逆转滋养细胞氧化应激损伤,其有望成为研究子痫前期发病机制的新靶点。

Objective To investigate the mechanism of SATB1 overexpression in protecting human trophoblast from oxidative stress injury. Methods Immunohistochemistry and Western blotting were used to determine the expression of SATB1 in placental tissue. Construct the overexpression lentivirus vectors of SATB1（LV-SATB1） and negative control vectors（LV- NC）. Cell treatment and classification of each group were as follows： normal culture group, H/R culture group, LV-SATB1＋ H/R culture group, LV-NC＋ H/R culture group. The expression and localization of SATB1 were detected by WB and indirect immunofluorescence. Flow cytometry was adopted to identify the level of reactive oxygen species（ROS） and the apoptotic index of cells. Changes of cell invasion rates were identified by transwell matrigel invasion assay. Results The level of SATB1 protein decreased in placental tissues of preeclamptic compared to that in normal third trimester（0.24±0.02 vs. 0.12±0.01, t=9.35,P〈0.05）. Transfected LV-SATB1 increased the expression of SATB1 notably in H/R group（0.92±0.17 vs. 0.58±0.15,P〈0.01）. The apoptosis index and accumulation of ROS in HTR8/SVneo cells induced by H/R could be reversed by transfected LV-SATB1 effectively（P〈0.01）. The invasion rates of cells decreased significantly in H/R group, while transfected LV-SATB1 could enhance the invasion rates in cells exposed to H/R（34.33±10.08 vs. 19.33±6.52,P〈0.05）. Conclusions Up-regulation of SATB1 can reverse the oxidative stress injury of trophoblast. SATB1 is expected to be the new target for the pathogenesis of preeclampsia.