缺氧诱导滋养细胞野生型p53诱导磷酸酶上调代偿凋亡的机制  

Compensatory role of wild-type p53-induced phosphatase in trophoblastic apoptosis in response to hypoxia

作　　者：谭彬[1] 童超[1] 袁雨[1] 林俐 漆洪波[1] Tan Bin;Tong Chao;Yuan Yu;Lin Li;Qi Hongbo(Department of Obstetrics,the First Affiliated Hospital of Chongqing Medical University,State Key Laboratory of Maternal and Fetal Medicine of Chongqing Municipality,International Collaborative Laboratory of Reproduction and Development of Chinese Ministry of Education,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院产科母胎医学重庆市重点实验室教育部母胎医学重点实验室,400016

出　　处：《中华围产医学杂志》2019年第10期712-721,共10页Chinese Journal of Perinatal Medicine

基　　金：国家重点研发计划(2016YFC1000407);国家自然科学基金(81520108013);高等学校学科创新引智计划(外专办发[2016]404);重庆市教委创新团队资助项目(CXTDX201601014).

摘　　要：目的探讨野生型p53诱导磷酸酶(wild-type p53-induced phosphatase, Wip1)依赖p53途径调控滋养细胞凋亡的机制,为子痫前期发病机制提供新的研究方向。方法收集2017年6月至2018年12月于重庆医科大学附属第一医院剖宫产分娩的正常孕妇(15例)与子痫前期孕妇(13例)的胎盘组织,收集早孕期(10例)人工流产者的绒毛和蜕膜组织;取绒毛外滋养细胞株HTR8/SVneo细胞,采用物理缺氧和化学缺氧方法进行缺氧培养。利用蛋白质印迹法和实时荧光定量聚合酶链反应分别检测胎盘组织中Wip1蛋白和mRNA水平;免疫组织化学方法和免疫荧光法检测Wip1组织细胞定位;采用病毒感染和缺氧干扰Wip1表达后,通过流式细胞技术检测细胞凋亡水平变化,蛋白质印迹法检测p53、磷酸化p53(phospho-p53, p-p53)、鼠双微同源体2(mouse double minute 2 homolog,Mdm2)和切割型含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase3, cl-cas3)凋亡通路相关分子的变化,并利用免疫共沉淀检测Wip1对Mdm2与p53相互作用的影响,并结合运用Mdm2-p53特异性抑制剂NVP-CGM097封闭结合位点进一步验证Wip1经Mdm2-p53途径调控滋养细胞凋亡。采用Student-t检验、Welch's-t检验和单因素方差分析进行统计学分析。结果(1)胎盘组织中Wip1主要表达于滋养细胞,在子痫前期胎盘(mRNA:1.711±0.141与0.860±0.126,t=4.496;蛋白:0.449±0.027与0.192±0.019,t=7.902)和物理缺氧(蛋白:1.376±0.086与0.977±0.114,t=2.792)、化学缺氧(蛋白:1.243±0.057与0.381±0.045,t=11.910)培养细胞中较相应对照组显著上调(P值均<0.05)。(2)与不影响Wip1表达的相应对照组比较,过表达Wip1可降低p53蛋白(物理缺氧:0.185±0.024与0.572±0.072;化学缺氧:0.400±0.067与0.803±0.064)、cl-cas3蛋白(物理缺氧:0.243±0.034与0.529±0.072;化学缺氧:0.179±0.011与0.368±0.025)和p-p53/53水平(物理缺氧:1.326±0.129与2.100±0.187;化学缺氧:0.473±0.028与0.925±0.036),降低细胞凋亡�Objective To investigate the mechanism of wild-type p53-induced phosphatase (Wip1) in regulating p53-dependent apoptosis of trophoblasts for further understanding the etiology of preeclampsia (PE).Methods Placenta tissues were collected from normal (n=15) and PE (n=13) gravidas who underwent caesarean section in the First Affiliated Hospital of Chongqing Medical University from June 2017 to December 2018. Chorionic villus and decidua tissues were collected from another 10 women who aborted in early pregnancy. Two in vitro trophoblastic hypoxia cultures were established by subjecting human chorionic trophoblast cells (HTR8/Svneo) to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 expressions at the transcriptional and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The localization of Wip1 in placental tissues and HTR8/Svneo cells was determined by immunohistochemistry and immunofluorescence. Cell apoptosis was assessed by flow cytometry after viral infection and hypoxia. And the changes of pathway-related molecules including p53, phospho-p53 (p-p53), mouse double minute 2 homolog (Mdm2) and cleaved caspase3 (cl-cas3) were measured by Western blotting. The impact of Wip1 on Mdm2-p53 interaction was examined by co-immunoprecipitation. NVP-CGM097, an Mdm2-p53 specific inhibitor, was administered in PE cell models to verify the regulation of Wip1 on trophoblastic apoptosis through Mdm2-p53 pathway. Independent student's t-test, Welch's t-test and one-way analysis of variance were used as statistical methods.Results(1) Wip1 expression, which was mainly in trophoblast cells, was significantly elevated in human PE placentas (mRNA: 1.711±0.141 vs 0.860±0.126, t=4.496;protein: 0.449±0.027 vs 0.192±0.019, t=7.902) and in both in vitro trophoblastic PE models (protein in HII: 1.376±0.086 vs 0.977±0.114, t=2.792;SIB: 1.243±0.057 vs 0.381±0.045, t=11.910) compared with the corresponding control groups (all P<

关 键 词：先兆子痫 细胞低氧 滋养层 蛋白质磷酸酶2C 细胞凋亡 

分 类 号：R714[医药卫生—妇产科学]