生殖医学杂志

目的探讨抗心磷脂抗体(ACA)阳性复发性流产小鼠胎盘组织中蛋白的表达谱特征并进行生物信息学分析。方法将20只雌性BALB/c小鼠随机分为正常对照组和ACA阳性流产模型组,每组10只。模型组于第1天和第8天通过腹腔注射β2-糖蛋白I(β2-GPI)建立ACA阳性流产模型,正常对照组于相同时间注射等体积生理盐水,腹腔注射后第18天,雌雄1∶1合笼,观察并记录妊娠时间。妊娠第15天处死小鼠,测量胎鼠、胎盘重量,计算胚胎丢失率,ELISA法检测血清、胎盘中ACA含量。采用Label-free蛋白质组学方法筛选两组小鼠胎盘组织中的差异蛋白,并进行GO富集、KEGG通路以及蛋白相互作网络分析,选择差异蛋白中的低氧诱导因子-1α(HIF-1α)、Toll样受体4(TLR-4)、凝血酶敏感蛋白-1(TSP-1)、T细胞免疫球蛋白黏蛋白分子-3(Tim-3)以及维甲酸相关孤核受体γt(RORγt)进行Western blot验证。结果与正常对照组相比,ACA阳性流产模型组胎鼠及胎盘重量显著下降(P<0.05),胚胎丢失率显著上升(P<0.05),血清、胎盘中ACA含量显著升高(P<0.05)。与正常对照组相比,ACA阳性流产小鼠胎盘组织中鉴别出87个差异表达蛋白,其中35个表达上调,52个表达下调;GO富集分析结果显示差异蛋白主要定位于细胞外,参与跨膜转运活性的调控;KEGG通路分析结果表明差异蛋白主要富集在代谢和磷脂酶D信号通路;蛋白相互作用分析发现差异蛋白中39个蛋白存在相互作用。Western blot验证结果显示,HIF-1α、TLR-4、TSP-1、Tim-3和RORγt表达量在模型组均显著上升(P<0.05),与蛋白组学结果一致。结论成功建立ACA阳性流产小鼠模型。ACA阳性流产小鼠胎盘组织中蛋白表达谱发生显著改变,差异蛋白主要与跨膜转运和代谢通路有关,这些蛋白的差异表达可能与ACA阳性流产有关。

Objective:To analyze the protein expression profiles and bioinformatics in the placenta of mice with anticardiolipin antibody(ACA) positive recurrent abortion.Methods:Twenty female BALB/c mice were randomly divided into normal control group and ACA positive abortion model group with 10 mice in each group. The mouse model of ACA positive abortion was established by injecting intraperitoneally with 2-glycoprotein I(β2-GPI) on the first and eighth days,and the normal group was injected with equal volume of normal saline at the same time. On the 18^th day after intraperitoneal injection,the male and female cages were 1∶1,and the pregnancy time was observed and recorded. On the 15^th day of pregnancy,the mice were killed,the weight of fetus and placenta were measured,and the embryo loss rate was calculated. The ACA level in placenta was detected by ELISA. The differentially expressed proteins(DEPs) in the placentas of mice in each group were screened by using Label-free proteomics and Go enrichment,KEGG pathway and protein interaction network analysis were performed. Hypoxia inducible factor-1 α(HIF-1 α),toll like receptor 4(TLR-4),thrombin sensitive protein-1(TSP-1),T cell immunoglobulin mucin molecule-3(Tim-3) and retinoic acid related orphan receptor gamma t(ROR γt) were selected for Western blot validation.Results:Compared with normal control mice,the weights of fetus and placenta were decreased,the embryo loss rate was increased and the ACA level in serum and placenta were increased. Compared with normal control mice,87 DEPs were identified in ACA positive mouse placenta,35 of which were significantly up-regulated and 52 significantly down-regulated. Go analysis showed that the DEPs were mainly located extracellular region and involved in the regulation of transmembrane transport activity. KEGG analysis showed that the DEPs were mainly concentrated in metabolism and phospholipase D signaling pathways. Interaction analysis showed that 39 DEPs interacted with each other. The results of Western blot were consistent with those of proteomics.Conclusions:The mouse model of ACA positive abortion was established successfully. The protein expression profiles in the placenta of ACA positive abortion mice were significantly changed. The differential expression proteins are related with transmembrane transport and metabolic pathway. Therefore,it is speculated that the differential expression of these proteins may be the causes of ACA positive abortion.