本文選題：組織學(xué)絨毛膜羊膜炎 + ox-AAT�。� 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：胎膜早破（premature rupture of membrane, PROM）即臨產(chǎn)前發(fā)生胎膜破裂。是產(chǎn)科常見的妊娠期并發(fā)癥，且近年發(fā)病呈上升趨勢。關(guān)于PROM的發(fā)病原因及發(fā)病機(jī)制，目前還未完全清楚。研究發(fā)現(xiàn)PROM與感染密切相關(guān)[1]。PROM合并感染后根據(jù)有無臨床表現(xiàn)，可分為臨床絨毛膜羊膜炎和亞臨床感染，亞臨床感染即組織學(xué)絨毛膜羊膜炎（HCA）。HCA早期無明顯臨床癥狀和體征，但分娩后胎盤組織的病理結(jié)果提示已有炎癥浸潤。PROM合并HCA可導(dǎo)致嚴(yán)重的母兒并發(fā)癥，甚至死亡。 近年來，早期診斷HCA受到學(xué)者的廣泛關(guān)注，但至今尚未找到敏感性、特異性強(qiáng)的指標(biāo)。 氧化型α1-抗胰蛋白酶（ox-AAT）是α1-抗胰蛋白酶（AAT）的一種存在形式。ox-AAT是炎癥反應(yīng)的產(chǎn)物，在加重、放大炎癥反應(yīng)的過程中起重要作用。炎癥細(xì)胞釋放的自由基可以作用AAT的活化位點(diǎn)，使其氧化成ox-AAT，從而失去抑制蛋白酶及抗炎功能。中性粒細(xì)胞彈性蛋白酶（NE），主要由中性粒細(xì)胞（PMN）脫顆粒釋放，是絲氨酸蛋白酶超家族成員之一。當(dāng)炎癥發(fā)生時(shí)，PMN發(fā)生趨化作用，可以清除病原菌，促進(jìn)損傷組織的修復(fù)。但是，炎癥反應(yīng)過度可導(dǎo)致NE釋放增多，發(fā)生組織的損傷及炎癥的放大現(xiàn)象。 目的 本研究通過檢測PROM孕婦血清、臍血血清及胎膜組織中ox-AAT和NE的水平及它們之間的相關(guān)性，探討ox-AAT、NE與組織學(xué)絨毛膜羊膜炎的關(guān)系，以期尋找早期診斷組織學(xué)絨毛膜羊膜炎的敏感指標(biāo)。 資料與方法 1研究對象與分組 選擇2012年9月至2013年3月在鄭州大學(xué)第三附屬醫(yī)院住院分娩的PROM孕婦，排除診斷為臨床絨毛膜羊膜炎、肝炎、腎炎、上呼吸道感染等感染性疾病的患者，共入選60例PROM孕婦。根據(jù)分娩后胎盤組織病理結(jié)果是否診斷為HCA分為兩組：①HCA組22例，②對照組38例。 PROM的診斷標(biāo)準(zhǔn)參照謝幸、茍文麗主編的《婦產(chǎn)科學(xué)》第8版[1]；臨床絨毛膜羊膜炎的診斷標(biāo)準(zhǔn)：孕婦T37.8℃、P120次/分、胎兒HR160次/分，，并伴下列兩條標(biāo)準(zhǔn)中的任意一條：子宮壓痛或羊水有臭味[2]；組織學(xué)絨毛膜羊膜炎（HCA）的診斷標(biāo)準(zhǔn)：胎盤組織HE染色后，根據(jù)顯微鏡下每高倍視野炎性細(xì)胞數(shù)量5個(gè)[3]診斷。 所有孕婦入院后記錄一般資料，檢測C反應(yīng)蛋白（CRP）及白細(xì)胞（WBC）計(jì)數(shù)。收取標(biāo)本前均取得入選對象的知情同意。 2實(shí)驗(yàn)方法 2.1血清標(biāo)本采集：所有入選對象入院后、應(yīng)用抗生素前，留取外周血5ml，離心后，取血清低溫保存。胎兒娩出后留取臍血5ml，離心低溫保存。 2.2胎膜、胎盤組織標(biāo)本采集：胎盤娩出后取胎膜破口至胎盤邊緣的胎膜，約1cm×4cm，冷生理鹽水沖洗表面血跡，使成卷狀，用大頭針固定，分為兩部分，一部分放入凍存管后迅速投入液氮，一部分甲醛固定。避開鈣化點(diǎn)取2cm×2cm大小胎盤全層3～4塊，甲醛固定。 2.3酶聯(lián)免疫吸附（enzyme-linked immunoadsordent assay，ELISA）實(shí)驗(yàn)檢測孕婦外周血、臍血中ox-AAT和NE的水平。 2.4采用逆轉(zhuǎn)錄PCR技術(shù)檢測胎膜組織中ox-AAT mRNA、NE mRNA的表達(dá)水平。 2.5采用免疫組化方法檢測ox-AAT、NE在胎膜、胎盤組織中的表達(dá)。 3統(tǒng)計(jì)學(xué)方法 應(yīng)用SPSS13.0統(tǒng)計(jì)軟件分析，計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差（X S）表示；組間比較采用獨(dú)立樣本的t檢驗(yàn)或秩和檢驗(yàn)。計(jì)數(shù)資料采用率表示，組間比較用2檢驗(yàn)。相關(guān)性分析采用Pearson相關(guān)分析。以α=0.05為檢驗(yàn)水準(zhǔn)。 結(jié)果 1HCA組和對照組一般資料的比較 兩組孕婦的年齡、分娩孕周比較，差異均無統(tǒng)計(jì)學(xué)意義（P0.05）；HCA組孕婦破膜-胎兒娩出時(shí)間、WBC計(jì)數(shù)及CRP水平較對照組高，但差異均無統(tǒng)計(jì)學(xué)意義（P0.05）。 2HCA組和對照組孕婦外周血、臍血中ox-AAT及NE的表達(dá)水平 HCA組孕婦血清中ox-AAT和NE的水平分別為（2.328±0.014）、（0.407±0.082）ng/L，對照組ox-AAT和NE的水平分別為（1.503±0.12）、（0.260±0.090）ng/L，HCA組與對照組比較，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）；HCA組臍血中ox-AAT和NE的水平分別為（2.920±0.182）、（0.744±0.065）ng/L，對照組ox-AAT和NE的水平分別為（2.433±0.004）、（0.415±0.010）ng/L，HCA組與對照組比較，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 3HCA組與對照組胎膜組織中ox-AAT mRNA、NE mRNA的表達(dá)水平 HCA組胎膜組織中ox-AAT mRNA的水平（0.960±0.03）和NE mRNA的水平（0.670±0.075）明顯高于對照組（0.532±0.01、0.271±0.09）水平，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 4HCA組與對照組胎膜組織中ox-AAT、NE蛋白水平的表達(dá) HCA組胎膜組織中ox-AAT的水平（0.023±0.005）和NE的水平（0.019±0.002）均高于對照組（0.015±0.002、0.005±0.001），差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 5HCA組與對照組胎盤組織中ox-AAT、NE蛋白水平的表達(dá) HCA組胎盤組織中ox-AAT的水平（0.182±0.001）和NE的水平（0.261±0.003）均顯著高于對照組（0.04±0.008、0.055±0.009），差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 6相關(guān)性分析 6.1兩組孕婦外周血中ox-AAT、NE的水平分別與臍血中兩者水平的相關(guān)性HCA組孕婦外周血中ox-AAT的水平與臍血中ox-AAT的水平呈正相關(guān)，r=0.825，P0.05；HCA組孕婦外周血中NE的水平與臍血中NE的水平呈正相關(guān)，r=0.847，P0.05。對照組分別無相關(guān)性。 6.2HCA組孕婦外周血中ox-AAT、NE的表達(dá)水平分別與胎膜組織中兩者水平的相關(guān)性 HCA組孕婦外周血中ox-AAT的表達(dá)水平與胎膜組織中ox-AAT的表達(dá)呈正相關(guān)性，r=0.836，P0.05；HCA組孕婦外周血中NE的表達(dá)水平與胎膜組織中NE的表達(dá)呈正相關(guān)性，r=0.867，P0.05。 6.3HCA組ox-AAT與NE的相關(guān)性 HCA組孕婦外周血中ox-AAT與NE呈正相關(guān)性，r=0.885，P0.05；臍血中ox-AAT與NE呈正相關(guān)性，r=0.756，P0.05；胎膜組織中ox-AAT和NE呈正相關(guān)性，r=0.876，P0.05。 7兩組孕婦及新生兒結(jié)局的比較 60例孕婦中無一例發(fā)生胎盤早剝、產(chǎn)后出血及產(chǎn)褥感染。HCA組以剖宮產(chǎn)終止妊娠者18例，對照組9例，組間比較，差異有統(tǒng)計(jì)學(xué)意義（2=12.59，P0.05）。60例新生兒，HCA組中1例因敗血癥死亡，15例發(fā)生肺炎，無新生兒臍炎及壞死性腸炎發(fā)生，對照組中13例發(fā)生肺炎，組間比較差異有統(tǒng)計(jì)學(xué)意義（2=6.461，P0.05）。 結(jié)論 1．孕婦外周血、臍血和胎膜組織中ox-AAT、NE的水平升高與組織學(xué)絨毛膜羊膜炎密切相關(guān)。 2．監(jiān)測血清中ox-AAT、NE的水平為組織學(xué)絨毛膜羊膜炎的早期診斷提供依據(jù)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714.433

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