胎膜早破孕妇外周血、脐血和胎膜组织中ox-AAT、NE的表达与组织学绒毛膜羊膜炎的关系

发布时间：2018-05-26 14:23

本文选题：组织学绒毛膜羊膜炎 + ox-AAT　；参考：《郑州大学》2014年硕士论文

【摘要】：胎膜早破（premature rupture of membrane, PROM）即临产前发生胎膜破裂。是产科常见的妊娠期并发症，且近年发病呈上升趋势。关于PROM的发病原因及发病机制，目前还未完全清楚。研究发现PROM与感染密切相关[1]。PROM合并感染后根据有无临床表现，可分为临床绒毛膜羊膜炎和亚临床感染，亚临床感染即组织学绒毛膜羊膜炎（HCA）。HCA早期无明显临床症状和体征，但分娩后胎盘组织的病理结果提示已有炎症浸润。PROM合并HCA可导致严重的母儿并发症，甚至死亡。近年来，早期诊断HCA受到学者的广泛关注，但至今尚未找到敏感性、特异性强的指标。氧化型α1-抗胰蛋白酶（ox-AAT）是α1-抗胰蛋白酶（AAT）的一种存在形式。ox-AAT是炎症反应的产物，在加重、放大炎症反应的过程中起重要作用。炎症细胞释放的自由基可以作用AAT的活化位点，使其氧化成ox-AAT，从而失去抑制蛋白酶及抗炎功能。中性粒细胞弹性蛋白酶（NE），主要由中性粒细胞（PMN）脱颗粒释放，是丝氨酸蛋白酶超家族成员之一。当炎症发生时，PMN发生趋化作用，可以清除病原菌，促进损伤组织的修复。但是，炎症反应过度可导致NE释放增多，发生组织的损伤及炎症的放大现象。目的本研究通过检测PROM孕妇血清、脐血血清及胎膜组织中ox-AAT和NE的水平及它们之间的相关性，探讨ox-AAT、NE与组织学绒毛膜羊膜炎的关系，以期寻找早期诊断组织学绒毛膜羊膜炎的敏感指标。资料与方法 1研究对象与分组选择2012年9月至2013年3月在郑州大学第三附属医院住院分娩的PROM孕妇，排除诊断为临床绒毛膜羊膜炎、肝炎、肾炎、上呼吸道感染等感染性疾病的患者，共入选60例PROM孕妇。根据分娩后胎盘组织病理结果是否诊断为HCA分为两组：①HCA组22例，②对照组38例。 PROM的诊断标准参照谢幸、苟文丽主编的《妇产科学》第8版[1]；临床绒毛膜羊膜炎的诊断标准：孕妇T37.8℃、P120次/分、胎儿HR160次/分，，并伴下列两条标准中的任意一条：子宫压痛或羊水有臭味[2]；组织学绒毛膜羊膜炎（HCA）的诊断标准：胎盘组织HE染色后，根据显微镜下每高倍视野炎性细胞数量5个[3]诊断。所有孕妇入院后记录一般资料，检测C反应蛋白（CRP）及白细胞（WBC）计数。收取标本前均取得入选对象的知情同意。 2实验方法 2.1血清标本采集：所有入选对象入院后、应用抗生素前，留取外周血5ml，离心后，取血清低温保存。胎儿娩出后留取脐血5ml，离心低温保存。 2.2胎膜、胎盘组织标本采集：胎盘娩出后取胎膜破口至胎盘边缘的胎膜，约1cm×4cm，冷生理盐水冲洗表面血迹，使成卷状，用大头针固定，分为两部分，一部分放入冻存管后迅速投入液氮，一部分甲醛固定。避开钙化点取2cm×2cm大小胎盘全层3～4块，甲醛固定。 2.3酶联免疫吸附（enzyme-linked immunoadsordent assay，ELISA）实验检测孕妇外周血、脐血中ox-AAT和NE的水平。 2.4采用逆转录PCR技术检测胎膜组织中ox-AAT mRNA、NE mRNA的表达水平。 2.5采用免疫组化方法检测ox-AAT、NE在胎膜、胎盘组织中的表达。 3统计学方法应用SPSS13.0统计软件分析，计量资料采用均数±标准差（X S）表示；组间比较采用独立样本的t检验或秩和检验。计数资料采用率表示，组间比较用2检验。相关性分析采用Pearson相关分析。以α=0.05为检验水准。结果 1HCA组和对照组一般资料的比较两组孕妇的年龄、分娩孕周比较，差异均无统计学意义（P0.05）；HCA组孕妇破膜-胎儿娩出时间、WBC计数及CRP水平较对照组高，但差异均无统计学意义（P0.05）。 2HCA组和对照组孕妇外周血、脐血中ox-AAT及NE的表达水平 HCA组孕妇血清中ox-AAT和NE的水平分别为（2.328±0.014）、（0.407±0.082）ng/L，对照组ox-AAT和NE的水平分别为（1.503±0.12）、（0.260±0.090）ng/L，HCA组与对照组比较，差异均有统计学意义（P0.05）；HCA组脐血中ox-AAT和NE的水平分别为（2.920±0.182）、（0.744±0.065）ng/L，对照组ox-AAT和NE的水平分别为（2.433±0.004）、（0.415±0.010）ng/L，HCA组与对照组比较，差异均有统计学意义（P0.05）。 3HCA组与对照组胎膜组织中ox-AAT mRNA、NE mRNA的表达水平 HCA组胎膜组织中ox-AAT mRNA的水平（0.960±0.03）和NE mRNA的水平（0.670±0.075）明显高于对照组（0.532±0.01、0.271±0.09）水平，差异均有统计学意义（P0.05）。 4HCA组与对照组胎膜组织中ox-AAT、NE蛋白水平的表达 HCA组胎膜组织中ox-AAT的水平（0.023±0.005）和NE的水平（0.019±0.002）均高于对照组（0.015±0.002、0.005±0.001），差异均有统计学意义（P0.05）。 5HCA组与对照组胎盘组织中ox-AAT、NE蛋白水平的表达 HCA组胎盘组织中ox-AAT的水平（0.182±0.001）和NE的水平（0.261±0.003）均显著高于对照组（0.04±0.008、0.055±0.009），差异均有统计学意义（P0.05）。 6相关性分析 6.1两组孕妇外周血中ox-AAT、NE的水平分别与脐血中两者水平的相关性HCA组孕妇外周血中ox-AAT的水平与脐血中ox-AAT的水平呈正相关，r=0.825，P0.05；HCA组孕妇外周血中NE的水平与脐血中NE的水平呈正相关，r=0.847，P0.05。对照组分别无相关性。 6.2HCA组孕妇外周血中ox-AAT、NE的表达水平分别与胎膜组织中两者水平的相关性 HCA组孕妇外周血中ox-AAT的表达水平与胎膜组织中ox-AAT的表达呈正相关性，r=0.836，P0.05；HCA组孕妇外周血中NE的表达水平与胎膜组织中NE的表达呈正相关性，r=0.867，P0.05。 6.3HCA组ox-AAT与NE的相关性 HCA组孕妇外周血中ox-AAT与NE呈正相关性，r=0.885，P0.05；脐血中ox-AAT与NE呈正相关性，r=0.756，P0.05；胎膜组织中ox-AAT和NE呈正相关性，r=0.876，P0.05。 7两组孕妇及新生儿结局的比较 60例孕妇中无一例发生胎盘早剥、产后出血及产褥感染。HCA组以剖宫产终止妊娠者18例，对照组9例，组间比较，差异有统计学意义（2=12.59，P0.05）。60例新生儿，HCA组中1例因败血症死亡，15例发生肺炎，无新生儿脐炎及坏死性肠炎发生，对照组中13例发生肺炎，组间比较差异有统计学意义（2=6.461，P0.05）。结论 1．孕妇外周血、脐血和胎膜组织中ox-AAT、NE的水平升高与组织学绒毛膜羊膜炎密切相关。 2．监测血清中ox-AAT、NE的水平为组织学绒毛膜羊膜炎的早期诊断提供依据。
[Abstract]:Premature rupture of the membranes (premature rupture of membrane, PROM) is the rupture of the membranes before parturient. It is a common pregnancy complication in obstetrics, and the incidence is on the rise in recent years. The etiology and pathogenesis of PROM are not completely clear. The study found that PROM and infection are closely related to infection after [1].PROM combined infection, based on the clinical manifestations, It can be divided into clinical chorioamnionitis and subclinical infection. Subclinical infection, namely histologic chorioamnionitis (HCA), has no obvious clinical symptoms and signs in the early stage of.HCA, but the pathological results of placental tissue after delivery suggest that inflammatory infiltration of.PROM and HCA can lead to severe mother and child onset, or even death.
In recent years, early diagnosis of HCA has attracted wide attention from scholars, but so far, no sensitive and specific index has yet been found.
Oxidized alpha 1- antitrypsin (ox-AAT) is a form of the existence form of alpha 1- antitrypsin (AAT), a form of.Ox-AAT, a product of inflammation, which plays an important role in aggravating and amplifying the inflammatory response. The free radicals released by inflammatory cells can act as the activation sites of AAT and oxidize them to ox-AAT, thereby losing the inhibition of protease and anti-inflammatory function. Neutrophil elastase (NE), which is released mainly by neutrophils (PMN), is one of the members of the serine protease superfamily. When inflammation occurs, the chemotactic effect of PMN can clear the pathogenic bacteria and promote the repair of damaged tissues. However, excessive inflammation can lead to the increase in the release of NE, the injury of tissue and the release of inflammation. Big phenomenon.
objective
To explore the relationship between the levels of ox-AAT and NE in the serum of PROM pregnant women, umbilical blood serum and fetal membranes and their correlation, the relationship between ox-AAT, NE and histology amnionitis was explored in order to find a sensitive index for early diagnosis of histopathology of chorionic amnionitis.
Information and methods
1 research objects and groups
PROM pregnant women who were hospitalized in Third Affiliated Hospital of Zhengzhou University from September 2012 to March 2013 were selected to exclude patients with infectious diseases such as chorioamnionitis, hepatitis, nephritis, and upper respiratory infection, and 60 cases of PROM pregnant women were selected. Two groups were divided into two groups according to the diagnosis of placental tissue disease after delivery: (1) group HCA 22 For example, 38 cases in the control group.
The diagnostic standard of PROM is referred to as Xie Li, the eighth edition of Obstetrics and Gynecology [1], go Wen Li's chief editor, clinical chorioamnionitis diagnostic criteria: pregnant women T37.8, P120 / sub, fetal HR160 / sub, and with any of the following two criteria: uterine pressure pain or amniotic fluid stink [2]; histology of chorionic amnionitis (HCA) diagnostic criteria: fetal fetal (HCA) After disc tissue HE staining, 5 [3] were diagnosed according to the number of inflammatory cells in every high magnification field under microscope.
All the pregnant women recorded general information after admission, detected C reactive protein (CRP) and white blood cell count (WBC). The informed consent was obtained before the specimen was collected.
2 experimental method
2.1 collection of serum specimens: after all the selected subjects were admitted to hospital, the peripheral blood 5ml was left before the application of antibiotics. After centrifugation, the serum was preserved at low temperature. After the birth of the fetus, the umbilical blood was retained for 5ml, and the centrifuge was kept at low temperature.
2.2 fetal membrane, placental tissue specimen collection: after delivery of placenta, the fetal membrane of fetal membrane break to the edge of placenta is taken, about 1cm x 4cm, cold physiological saline rinse the surface blood, so that it is curled, fixed with a pin and divided into two parts. A part of the cryopreservation tube is put into liquid nitrogen quickly and a part of formaldehyde is fixed. Avoid calcification point to take the whole layer of 2cm * 2cm placenta 3 To 4 blocks, formaldehyde is fixed.
2.3 enzyme-linked immunoadsordent assay (ELISA) was used to detect the levels of ox-AAT and NE in peripheral blood and umbilical cord blood of pregnant women.
2.4 reverse transcription PCR was used to detect the expression level of ox-AAT mRNA and NE mRNA in fetal membranes.
2.5 immunohistochemical method was used to detect the expression of ox-AAT and NE in fetal membranes and placenta tissues.
3 statistical method
Using SPSS13.0 statistical software analysis, the measurement data were represented by mean number + standard deviation (X S); the group was compared with the independent sample of t test or rank sum test. The ratio of counting data was expressed, the group was compared with 2 test. The correlation analysis was analyzed by Pearson correlation analysis. Alpha =0.05 was used as the test standard.
Result
Comparison of general data between group 1HCA and control group
There was no significant difference in the age of two groups of pregnant women and the comparison of pregnancy weeks in childbirth (P0.05), while in group HCA, the time of rupture of the fetus, the time of delivery of the fetus, the count of WBC and the level of CRP were higher than those of the control group, but the difference was not statistically significant (P0.05).
Expression levels of ox-AAT and NE in peripheral blood and cord blood of pregnant women in 2HCA and control groups
The level of ox-AAT and NE in the serum of HCA group was (2.328 + 0.014), (0.407 + 0.082) ng/L, and the level of ox-AAT and NE in the control group was (1.503 + 0.12) and (0.260 + 0.090) ng/L respectively. The difference was statistically significant (P0.05) compared with the control group (P0.05), and the level of ox-AAT and NE in the HCA group was (2.920 + 0.182) and (0.744 + 0.065), respectively. The levels of ox-AAT and NE were respectively (2.433 + 0.004), (0.415 + 0.010) ng/L, and the difference between HCA group and control group was statistically significant (P0.05).
Expression levels of ox-AAT mRNA, NE mRNA in fetal membranes of 3HCA group and control group
The level of ox-AAT mRNA (0.960 + 0.03) and NE mRNA (0.670 + 0.075) in the fetal membrane tissues (0.670 + 0.075) were significantly higher than those of the control group (0.532 + 0.01,0.271 + 0.09), and the difference was statistically significant (P0.05).
Expression of ox-AAT and NE protein in fetal membranes of 4HCA group and control group
The level of ox-AAT (0.023 + 0.005) and NE (0.019 + 0.002) in the HCA group were higher than those in the control group (0.015 + 0.002,0.005 + 0.001), and the difference was statistically significant (P0.05).
Expression of ox-AAT and NE protein in placenta of 5HCA and control groups
The level of ox-AAT (0.182 + 0.001) and NE (0.261 + 0.003) in the placental tissue of group HCA were significantly higher than those in the control group (0.04 + 0.008,0.055 + 0.009), and the difference was statistically significant (P0.05).
6 correlation analysis
6.1 the levels of ox-AAT, NE in the peripheral blood of the two groups of pregnant women were correlated with the levels of umbilical blood respectively. The level of ox-AAT in the peripheral blood of the pregnant women was positively correlated with the level of ox-AAT in the umbilical blood, r=0.825, P0.05; the level of NE in peripheral blood of the pregnant women of group HCA was not related to the level of Cheng Zhengxiang, r=0.847, and P0.05. control in the umbilical blood.
The levels of ox-AAT and NE in peripheral blood of pregnant women in group 6.2HCA were correlated with the levels of fetal membranes.
The expression level of ox-AAT in peripheral blood of HCA group was positively correlated with the expression of ox-AAT in fetal membrane, r=0.836, P0.05; the expression level of NE in peripheral blood of pregnant women in group HCA was positively correlated with the expression of NE in fetal membrane, r=0.867, P0.05..
Correlation between ox-AAT and NE in group 6.3HCA
In group HCA, there was a positive correlation between ox-AAT and NE in the peripheral blood of pregnant women, r=0.885, P0.05, and the positive correlation between ox-AAT and NE in umbilical cord blood, r=0.756, P0.05, ox-AAT and NE in the fetal membrane, r=0.876, and NE.
7 Comparison between two groups of pregnant women and newborns
Among the 60 pregnant women, there were no placental abruption, 18 postpartum hemorrhage and puerperal infection in.HCA group, 18 cases of cesarean section and 9 cases in control group. The difference was statistically significant (2=12.59, P0.05).60 newborns, 15 cases of septicemia in group HCA, 15 cases of pulmonary inflammation, no neonatal oculitis and necrotizing enteritis, 13 in the control group. There was a significant difference between the two groups (2=6.461, P0.05).
conclusion
1. the level of ox-AAT and NE in peripheral blood, umbilical cord blood and fetal membranes of pregnant women is closely related to histologic chorioamnionitis.
2. monitoring serum levels of ox-AAT and NE can provide evidence for early diagnosis of histologic chorioamnionitis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.433

【参考文献】