非重度晚发型子痫前期患者血清中差异蛋白质与血管内皮损伤的关系

Relationship between serum proteins and vascular endothelial injury in patients with late-onset preeclampsia

目的探索并寻找晚发型子痫前期特异性蛋白质,在分子生物学水平上为该疾病的筛查及预防提供一定的实验依据。方法选择2016年7月至2017年3月在哈尔滨医科大学附属第四医院进行规律产前检查中非重度晚发型子痫前期孕妇30例(研究组),妊娠34周后血压≥18.6/12.0 k Pa(140/90 mm Hg),妊娠37~40+6周。同时,随机选择无任何并发症及合并症的正常妊娠孕妇30例(对照组),妊娠37~40+6周。动态观察孕妇直至妊娠足月住院分娩。在此阶段内采集孕妇血清。利用双向凝胶电泳技术(2DE),分别检测两组血清蛋白质,利用Image Master 2D platinum 5.0软件分析所得图像结果,寻找到特异性蛋白质标志物,并通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质,利用Mascot软件搜索NCBInr数据库寻找匹配的蛋白质。同时将改变差异蛋白质浓度的血清传代培养人血管内皮细胞,体外模拟晚发型子痫前期状态,建立血管内皮细胞损伤模型,用噻唑蓝(MTT)法测其增殖活性及组织纤溶酶原激活剂(t-PA)及纤溶酶原激活剂抑制剂(PAI)活性,并测其上清液中超氧化物歧化酶(SOD)活性及丙二醛(MDA)的含量。结果研究组与对照组血清蛋白质比较,共发现19种蛋白质存在差异表达,其中15种蛋白质表达下降,4种蛋白质表达升高。其中差异倍数最大的蛋白质为人α-胰蛋白酶重链H4(ITIH4),其表达含量下降最为明显。体外血管内皮细胞损伤模型实验,过氧化氢处理过的血管内皮细胞增殖活性明显低于正常组,加入不同浓度的ITIH4可以不同程度地提高其增殖活性,且当质量浓度为100 mg/L时,其增殖活性升高最为明显(P<0.01)。以t-PA及PAI间接反映血管内皮细胞损伤,经过氧化氢诱导过的血管内皮细胞的PAI活性较正常组明显升高(P<0.01),且t-PA活性明显降低(P<0.01)。当加入质量浓度为100 mg/L ITIH4后,t-PA活性增加最明显,PAI活性降低最明显(P<0.01)。结论血清中ITIH4表达量的下降,与血管内皮细胞损伤存在一定关系,故此改变可能参与了晚发型子痫前期的发生、发展。将为临床上筛查、预防子痫前期,奠定一定的实验基础。

Objective To explore and search for specific proteins of the late-onset preeclampsia, and provide basis for screening and prevention of the disease at molecular biology level. Methods A total of 30 non-severe late-onset preeclampsia pregnant women（study group） undergoing regular prenatal care at the Fourth Affiliated Hospital of Harbin Medical Uni-versity from July 2016 to March 2017, which blood pressure ≥ 18.6/12.0 k Pa（140/90 mm Hg） after 34-week gestation with37-40＋6 weeks＇ gestation were enrolled. At the same time, 30 normal pregnant women with 37-40＋6 weeks of gestation without any complications were enrolled as control group. All of the pregnant women were given dynamic observation until full-term pregnancy childbirth. Maternal serum were collected and two-dimensional gel electrophoresis technology（2 DE） was used to detect serum proteins of 2 groups and the images were analyzed by Image Master 2 D platinum 5.0 software. The results of the images obtained were searched for specific protein markers and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry（MALDI-TOF-MS）. Mascot software was used to search NCBInr database for matching proteins. At the same time, human vascular endothelial cell line was subcultured, and late-onset preeclampsia state was simulated in vitro by changing concentration of differential protein in serum. The injury model of vascular endothelial cells was established and MTT was used to measure its proliferation. Activity and tissue-type plasminogen activator（t-PA） and plasminogen activator inhibitor（PAI） activity were measured, and superoxide dismutase（SOD） activity and malondialdehyde in the supernatant were measured. Results Compared the serum protein between study group and control group, a total of 19 proteins were found to be differentially expressed, of which 15 proteins decreased and 4 proteins increased. Among them, the protein with the largest difference was human inter-alpha trypsin inhibitor heavy chain H4（ITIH4）, and its expression decreased most significantly. In vitro vascular endothelial cell injury model results showed that proliferation activity of hydrogen peroxide treated vascular endothelial cells was significantly lower than that of normal group. The addition of different concentrations of ITIH4 increased proliferative activity of the cells to various degrees, and concentration was 100 mg/L, the proliferation activity increased most significantly（P〈0.01）. The t-PA and PAI indirectly reflected the injury of vascular endothelial cells. The PAI activity of vascular endothelial cells induced by hydrogen peroxide was significantly higher than that of normal group（P〈0.01）,and t-PA activity was significantly decreased（P〈0.01）. With the addition of ITIH4 with the mass concentration of 100 mg/L,the activity of t-PA increased most significantly, and the activity of PAI decreased most significantly（P〈0.01）. Conclusion It is demonstrated that the decrease of ITIH4 expression in serum is related to the damage of vascular endothelial cells, so the change may be involved in occurrence and development of late-onset preeclampsia, which will provide certain experimental basis for clinical screening and prevention of preeclampsia.