本文選題：羊水栓塞　切入點(diǎn)：IL-6　出處：《遵義醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：通過建立羊水栓塞動物模型，分析外周血液動力學(xué)改變、測定血中IL-6、IL-8含量的變化，進(jìn)一步探索羊水栓塞的發(fā)病機(jī)制。 方法：1．構(gòu)建羊水栓塞的大鼠模型 正常懷孕大鼠30只，重300g~500g，胎齡20~25d。隨機(jī)分為3組：對照組（生理鹽水組）、實驗組1（羊水原液組）、實驗組2（羊水胎糞液組），每組為10只。待麻醉生效后，大鼠行子宮次全切除術(shù)后，關(guān)腹。分離孕鼠左頸總動脈，從左頸總動脈插管，并將其接通三通管連接于二通道生理記錄儀上，測出連續(xù)的血液動力學(xué)指標(biāo)。三組經(jīng)腹腔靜脈分別注入生理鹽水、羊水原液、羊水胎糞混合離心上清液，分別于注射前、注射后60min由左頸總動脈取血，量1ml（若在觀察過程中大鼠出現(xiàn)死亡立即取血），將血液離心10min（3000r/min），取其上清液放置于-20°C恒溫冰箱中保存?zhèn)溆茫糸_胸腔，取孕鼠右肺組織留做病理學(xué)檢查。 2．羊水胎糞上清液的制備 打開胎鼠腹壁，取出胎鼠大腸，用注射器抽取羊水沖洗胎鼠腸腔（或?qū)⑻ゼS輕輕擠壓出來），用勻漿器磨碎，配置成羊水中混有1%~7%胎糞的羊水胎糞混合懸液，然后離心10min （3000r/min），留取上清液。 3.IL-6的檢測方法 IL-6的檢測方法：用酶聯(lián)免疫吸附實驗（ELISA）對IL-6進(jìn)行檢測。將已知IL-6濃度的標(biāo)準(zhǔn)品、未知濃度的樣品加入微孔酶標(biāo)板內(nèi)進(jìn)行檢測。先將IL-6和生物素標(biāo)記的抗體同時溫育。洗滌后，加入親和素標(biāo)記過的HRP。再經(jīng)過溫育和洗滌，去除未結(jié)合的酶結(jié)合物，然后加入底物A、B，和酶結(jié)合物同時作用，產(chǎn)生顏色。顏色的深淺和樣品中IL-6的濃度呈比例關(guān)系。 4.IL-8的檢測方法 IL-8的檢測方法：用酶聯(lián)免疫吸附實驗（ELISA）對IL-8進(jìn)行檢。將已知IL-8濃度的標(biāo)準(zhǔn)品、未知濃度的樣品加入微孔酶標(biāo)板內(nèi)進(jìn)行檢測。先將IL-8和生物素標(biāo)記的抗體同時溫育。洗滌后，加入親和素標(biāo)記過的HRP。再經(jīng)過溫育和洗滌，去除未結(jié)合的酶結(jié)合物，然后加入底物A、B，和酶結(jié)合物同時作用。產(chǎn)生顏色。顏色的深淺和樣品中IL-8的濃度呈比例關(guān)系。 所有數(shù)據(jù)使用SPSS18.0處理，組內(nèi)采用t檢驗，組間比較用多個樣本均數(shù)間的兩兩比較。P0.05有統(tǒng)計學(xué)意義。 結(jié)果： 1.大鼠羊水栓塞模型建立成功，實驗組1、實驗組2孕鼠臨床表現(xiàn)比較嚴(yán)重，實驗組1孕鼠60min內(nèi)無死亡，實驗組2孕鼠60min內(nèi)1只死亡。三組孕鼠外周血液動力學(xué)結(jié)果：對照組血液動力學(xué)指標(biāo)有一過性改變，無統(tǒng)計學(xué)意義（P0.05），實驗組1、實驗組2與對照組動脈收縮壓、舒張壓及平均動脈壓相比有明顯下降（P0.05），以注入羊水后10min最為明顯，30min至60min維持低水平，未恢復(fù)正常，，實驗組2中有一只孕鼠出現(xiàn)平均動脈血壓持續(xù)性下降約至20mmHg發(fā)展成心衰，實驗組1與實驗組2之間差異不明顯，無統(tǒng)計學(xué)意義（P0.05），三組孕鼠心率改變差異無統(tǒng)計學(xué)意義（P0.05）。 2.IL-6濃度：對照組注射前(20.38±7.73)μg/ml，注射后(19.47±6.46)μg/ml；實驗組1注射前(21.41±6.47)μg/ml，注射后(30.74±5.61)μg/ml；實驗組2注射前(20.27±5.51)μg/ml，注射后(36.44±5.47)μg/ml。 3.IL-8濃度：對照組注射前(15.78±4.36)μg/ml，注射后(15.29±4.76)μg/ml；實驗組1注射前(16.21±3.89)μg/ml，注射后(23.02±8.06)μg/ml；實驗組2注射前(15.49±4.25)μg/ml，注射后(38.8±9.02)μg/ml。 4. HE染色顯示實驗組1與實驗組2可見不同程度的肺水腫，大量的炎性細(xì)胞浸潤，肺血管內(nèi)見鱗狀上皮、粘液等羊水成分，對照組無明顯改變。 5. CK10免疫組織化學(xué)染色檢測可見實驗組1與實驗組2在大鼠肺血管含有鱗狀上皮等羊水有形成分，對照組無明顯改變。 6. APM染色可見實驗組1與實驗組2肺血管里角化鱗狀上皮呈桃紅色、管腔中粘液呈藍(lán)色、中性粒細(xì)胞胞質(zhì)淡藍(lán)或無色核紅色，對照組無明顯改變。 結(jié)論： 1.孕鼠臨床表現(xiàn)的嚴(yán)重程度與進(jìn)入母體的羊水成分有關(guān)，出現(xiàn)胎糞污染時要警惕AFE。 2.實驗組IL-6含量增加，以羊水胎糞組增加更為明顯，說明胎糞里含有的某些物質(zhì)刺激機(jī)體引起IL-6大量激活。 3.實驗組IL-8含量增加，以羊水胎糞組增加更為明顯，說明胎糞里含有的某些物質(zhì)刺激機(jī)體引起IL-8大量激活。
[Abstract]:Objective: to establish an amniotic fluid embolism animal model and analyze the changes of peripheral blood dynamics, and to detect the changes of IL-6 and IL-8 contents in blood, and further explore the pathogenesis of amniotic fluid embolism.
Methods: 1. the rat model of amniotic fluid embolism was constructed.
Normal pregnant rats 30, weight 300g~500g, gestational age 20~25d. were randomly divided into 3 groups: control group (saline group), experimental group 1 (amniotic fluid solution group) experimental group, 2 (meconium fluid group), each group with 10 rats. After the anesthetic effect, the abdomen of rats underwent hysterectomy after resection. The left carotid artery from the separation of pregnant rats, left common carotid artery, and the three on a pipe connected to the two channel physiological recorder, measure the hemodynamic indexes of continuous. Three groups were injected with normal saline by intraperitoneal vein, amniotic fluid meconium mixed liquid, centrifugal supernatant, respectively before injection after injection, 60min from the left carotid artery blood, the amount of 1ml (if in the observation process rats died immediately take blood), blood 10min (3000r/min), centrifugation the supernatants were placed in -20 ~ C constant temperature refrigerator spare, cut open the chest, take the pregnant rat right lung tissue for pathology examination.
Preparation of 2. amniotic fluid meconium supernatant
Open the fetal abdominal wall, the fetus out of large intestine flushing of fetal rat intestinal cavity with injection syringe (or amniotic fluid meconium gently squeeze out), with homogenizer ground configured meconium suspension in amniotic fluid meconium mixed with 1%~7%, 10min (3000r/min), and then centrifuged to take the supernatant.
Detection methods of 3.IL-6
IL-6 detection methods: by enzyme-linked immunosorbent assay (ELISA) for the detection of IL-6. The standard of known concentration of IL-6, concentration of unknown samples by adding micro ELISA plate are detected. The IL-6 antibody and biotin labeled and incubated. After washing, adding streptavidin tagged HRP. after incubation and washing to remove unbound enzyme conjugate, and then adding substrate A, B, and enzyme conjugates. At the same time, color was proportional to the concentration of IL-6 and the color samples.
Detection methods of 4.IL-8
IL-8 detection methods: by enzyme-linked immunosorbent assay (ELISA) for detection of IL-8. The standard of known concentration of IL-8, concentration of unknown samples by adding micro ELISA plate are detected. The IL-8 antibody and biotin labeled and incubated. After washing, adding streptavidin tagged HRP. after incubation and washing to remove unbound enzyme conjugate, and then adding substrate A, B, and enzyme conjugates simultaneously. To produce color. The concentration of IL-8 was proportional to the depth of color and samples.
All data were treated with SPSS18.0, and t test was used in the group. The comparison between groups with 22 of multiple samples compared with.P0.05 was statistically significant.
Result錛

本文編號：1712882