IL-6、IL-8与羊水栓塞关系的实验研究

发布时间：2018-04-05 03:03

  本文选题：羊水栓塞　切入点：IL-6　出处：《遵义医学院》2014年硕士论文

【摘要】：目的：通过建立羊水栓塞动物模型，分析外周血液动力学改变、测定血中IL-6、IL-8含量的变化，进一步探索羊水栓塞的发病机制。 方法：1．构建羊水栓塞的大鼠模型 正常怀孕大鼠30只，重300g~500g，胎龄20~25d。随机分为3组：对照组（生理盐水组）、实验组1（羊水原液组）、实验组2（羊水胎粪液组），每组为10只。待麻醉生效后，大鼠行子宫次全切除术后，关腹。分离孕鼠左颈总动脉，从左颈总动脉插管，并将其接通三通管连接于二通道生理记录仪上，测出连续的血液动力学指标。三组经腹腔静脉分别注入生理盐水、羊水原液、羊水胎粪混合离心上清液，分别于注射前、注射后60min由左颈总动脉取血，量1ml（若在观察过程中大鼠出现死亡立即取血），将血液离心10min（3000r/min），取其上清液放置于-20°C恒温冰箱中保存备用，剪开胸腔，取孕鼠右肺组织留做病理学检查。 2．羊水胎粪上清液的制备 打开胎鼠腹壁，取出胎鼠大肠，用注射器抽取羊水冲洗胎鼠肠腔（或将胎粪轻轻挤压出来），用匀浆器磨碎，配置成羊水中混有1%~7%胎粪的羊水胎粪混合悬液，然后离心10min （3000r/min），留取上清液。 3.IL-6的检测方法 IL-6的检测方法：用酶联免疫吸附实验（ELISA）对IL-6进行检测。将已知IL-6浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。先将IL-6和生物素标记的抗体同时温育。洗涤后，加入亲和素标记过的HRP。再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用，产生颜色。颜色的深浅和样品中IL-6的浓度呈比例关系。 4.IL-8的检测方法 IL-8的检测方法：用酶联免疫吸附实验（ELISA）对IL-8进行检。将已知IL-8浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。先将IL-8和生物素标记的抗体同时温育。洗涤后，加入亲和素标记过的HRP。再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。产生颜色。颜色的深浅和样品中IL-8的浓度呈比例关系。 所有数据使用SPSS18.0处理，组内采用t检验，组间比较用多个样本均数间的两两比较。P0.05有统计学意义。 结果： 1.大鼠羊水栓塞模型建立成功，实验组1、实验组2孕鼠临床表现比较严重，实验组1孕鼠60min内无死亡，实验组2孕鼠60min内1只死亡。三组孕鼠外周血液动力学结果：对照组血液动力学指标有一过性改变，无统计学意义（P0.05），实验组1、实验组2与对照组动脉收缩压、舒张压及平均动脉压相比有明显下降（P0.05），以注入羊水后10min最为明显，30min至60min维持低水平，未恢复正常，，实验组2中有一只孕鼠出现平均动脉血压持续性下降约至20mmHg发展成心衰，实验组1与实验组2之间差异不明显，无统计学意义（P0.05），三组孕鼠心率改变差异无统计学意义（P0.05）。 2.IL-6浓度：对照组注射前(20.38±7.73)μg/ml，注射后(19.47±6.46)μg/ml；实验组1注射前(21.41±6.47)μg/ml，注射后(30.74±5.61)μg/ml；实验组2注射前(20.27±5.51)μg/ml，注射后(36.44±5.47)μg/ml。 3.IL-8浓度：对照组注射前(15.78±4.36)μg/ml，注射后(15.29±4.76)μg/ml；实验组1注射前(16.21±3.89)μg/ml，注射后(23.02±8.06)μg/ml；实验组2注射前(15.49±4.25)μg/ml，注射后(38.8±9.02)μg/ml。 4. HE染色显示实验组1与实验组2可见不同程度的肺水肿，大量的炎性细胞浸润，肺血管内见鳞状上皮、粘液等羊水成分，对照组无明显改变。 5. CK10免疫组织化学染色检测可见实验组1与实验组2在大鼠肺血管含有鳞状上皮等羊水有形成分，对照组无明显改变。 6. APM染色可见实验组1与实验组2肺血管里角化鳞状上皮呈桃红色、管腔中粘液呈蓝色、中性粒细胞胞质淡蓝或无色核红色，对照组无明显改变。 结论： 1.孕鼠临床表现的严重程度与进入母体的羊水成分有关，出现胎粪污染时要警惕AFE。 2.实验组IL-6含量增加，以羊水胎粪组增加更为明显，说明胎粪里含有的某些物质刺激机体引起IL-6大量激活。 3.实验组IL-8含量增加，以羊水胎粪组增加更为明显，说明胎粪里含有的某些物质刺激机体引起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锛

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