【作者】 张涛；

【导师】 孟金来；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2021， 硕士

【摘要】 研究背景:子痫前期是一种严重的妊娠期并发症,发病率可高达10%,严重威胁母婴安全。目前,子痫前期的发病机制尚不明确,螺旋动脉重构障碍致使滋养细胞侵袭不足,发育不良的胎盘释放损伤因子到母体外周循环,造成血管内皮细胞损伤和促进系统性炎症反应的激活,出现由血管病变引起的多器官疾病的子痫前期。外泌体是携带胞质蛋白、脂质与遗传物质的纳米囊泡,胎盘来源外泌体早在妊娠6周就在母体循环中被发现,介导胎盘与母体之间的细胞通讯。子痫前期患者外泌体的分泌、组分与活性有所改变,已有研究证实miR-320c在PE患者血清中表达减低,且有研究发现miR-320c在肿瘤细胞增殖、迁移中发挥作用。研究目的:研究子痫前期患者与正常孕妇外周血中外泌体分泌水平差异,以及病情严重程度对外泌体分泌的影响。结合相关临床检测指标,探究外泌体在子痫前期发病中发挥的作用;研究在子痫前期外泌体中miR-320c含量的改变,探究miR-320c对VEGF表达的影响,分析这一改变在其在子痫前期发病中可能的作用,为早期对疾病预测及发现新的治疗靶点提供依据。研究方法:本实验收集2020年6月至2021年3月省立医院就的住院患者60例,其中子痫前期患者30例,正常足月妊娠孕妇20例,正常早孕患者10例,收集外周血标本,超速离心法分离外泌体并进行粒径分布、形态学及标记物鉴定,BCA蛋白定量法测定其浓度,比较正常及子痫前期患者间、病情程度组间分泌水平的差异以及与孕周变化的相关性,结合临床检测指标分析其在妊娠及发病中的意义;实时荧光定量PCR检测外泌体中miR-320c表达差异,RT-PCR数据用2-△△CT法进行分析;利用western-blot方法检测蛋白表达,ImageJ进行western-blot条带灰度分析;使用GraphPad Prism 8.0软件处理实验数据并进行作图;用SPSS22.0软件统计学分析;两个独立样本间采用t检验;计量资料采用均数±标准差表示;相关性分析采用Pearson相关分析;P<0.05表示差异具有统计学意义。研究结果:1.超速离心法可从正常孕妇及子痫前期患者外周循环中分离到较高纯度外泌体;2.子痫前期(重度)患者外周循环外泌体分泌水平高于正常妊娠(P<0.05),而子痫前期(轻度)与正常足月妊娠间无统计学差异(P>0.05);子痫患者及正常妊娠晚期外周循环外泌体均较妊娠早期显著增加(P<0.001),但子痫前期(轻度)与子痫前期(重度)间分泌水平无差异(P>0.05);在早发型子痫前期中,其含量与疾病严重程度呈正相关(r=0.449,P=0.047),而晚发型子痫前期中,相关关系并不明确(r=0.552,P=0.122)3.研究发现外周血中外泌体分泌与孕妇体重指数(BMI)成正相关(r=0.424,P=0.001),与尿蛋白水平成正相关(r=0.602,P<0.001),与白蛋白水平呈负相关关系(r=-0.595,P<0.001),而与孕周、终止妊娠时机、年龄等因素无明显相关(P>0.05)。4.子痫前期患者外泌体miRNA-320c表达较正常妊娠减低(P<0.05),且与疾病严重程度成负相关(r=0.535,P<0.01),与尿蛋白水平成负相关(r=-0.745,P<0.001),与血清白蛋白水平为正相关(r=0.632,P<0.001);模拟物转染人脐静脉内皮细胞上调miR-320c表达后,VEGF蛋白表达明显减低(P<0.05)。结论:正常妊娠与重度子痫前期患者期外泌体水平存在差异,孕周增长和疾病加重均促进机体外泌体的分泌,且病理妊娠中外泌体组分有所改变。其中,本实验中miR-320c在子痫前期患者外泌体中表达减低,且上调HUVEC中miR-320c表达,VEGF蛋白的表达明显降低。证实子痫前期外泌体活性组分的改变可能在子痫前期患者全身血管内皮损伤的发生及炎症反应激活中发挥作用,机体合成、释放信号分子组分有所改变的大量活性囊泡,运送至损伤部位与受体靶细胞相互作用,在子痫前期血管损伤及修复、缺氧组织血管新生中发挥重要作用。更多还原

【Abstract】 Background:Preeclampsia(PE)is a severe gestational hypertension disease with a incidence of up to 10%,which seriously threatens the health of mother and child.At present,the pathogenesis of preeclampsia is not clear.Insufficient spiral artery remodeling leads to placental insufficiency,the dysplasia of the placenta releases injury factors to the maternal peripheral circulation,causing vascular endothelial cell damage and promoting the activation of systemic inflammatory response,leading to preeclampsia with multiple organ diseases caused by vascular lesions.Exosomes are nanoscale vesicles carrying cytoplasmic proteins,lipids,and genetic material.Placenta-derived exosomes are found in maternal circulation as early as 6 weeks of gestation,mediating cellular communication between the placenta and the mother.The secretion,composition and activity of exosomes in patients with preeclampsia are changed.Studies have confirmed that the expression of miR-320c is reduced in serum of PE patients,and some studies have found that miR-320c plays a role in the proliferation and migration of tumor cells.Objectives:To study the difference of exosome secretion levels in peripheral blood between preeclampsia patients and normal pregnant women,as well as the influence of the severity of the disease on exosome secretion,and to explore the role of exosomes in the pathogenesis of preeclampsia combined with relevant clinical detection indicators To study the changes of miR-320c content in exosomes of preeclampsia and explore the effect of miR-320c on VEGF expression,and analyze the possible role of this change in the pathogenesis of preeclampsia,so as to provide the basis for disease prediction and new therapeutic targets.Methods:This study collected in June 2020 to March 2021 provincial hospital,60 cases in hospitalized patients,preeclampsia patients,30 cases of normal full-term pregnancy pregnant women 20 cases,10 cases of normal pregnancy patients,collecting peripheral blood specimens,ultracentrifugation separation secrete body and to identify the particle size distribution,morphology and markers,BCA protein quantitative method to measure its concentration,To compare the difference of secretory level between normal and preeclampsia patients and between the groups with the severity of the disease and the correlation with the changes of gestational period,and to analyze the significance of the secretory level in pregnancy and morbidity combined with the clinical detection indexes.The expression difference of miR-320c in exosomes was detected by real-time fluorescence quantitative PCR,and the RT-PCR data were analyzed by 2-△△CT method.Western-blot method was used to detect the protein expression,and ImageJ was used for grey-scale analysis of the Western-blot bands.Graphpad Prism 8.0 software was used to process the experimental data and make a map;SPSS22.0 software was used for statistical analysis;T test was used between two independent samples.Measurement data were expressed as mean±standard deviation.Pearson correlation analysis was used for correlation analysis.A P value less than 0.05 indicated a statistically significant difference.Results:1.High purity exosomes can be isolated from peripheral circulation of normal pregnant women and preeclampsia patients by hypervelocity centrifugation;2.The secretion level of peripheral circulating exosomes in patients with severe preeclampsia was higher than that in normal pregnancy(P<0.05),and there was no significant difference between preeclampsia and full-term pregnancy(P>0.05);Circulating exosomes were significantly increased in patients with eclampsia and in normal third trimester(P<0.001),but there was no difference in secretion levels between preeclampsia and severe preeclampsia(P>0.05);In early onset preeclampsia,its content was positively correlated with disease severity(r=0.449,P=0.047),while in late onset preeclampsia,the correlation was not clear(r=0.552,P=0.122).3.The study found that peripheral blood secretion of exosomes was positively correlated with body mass index(BMI)(r=0.424,P=0.001)and urinary protein level(r=0.602,P<0.001),was negatively correlated with albumin level(r=-0.595,P<0.001),and there was no significant correlation between gestational age,time of termination of pregnancy,age and other factors(P<0.05).4.The expression of miRNA-320c in exosomes of preeclampsia patients was lower than that of normal pregnancy(P<0.05)and was negatively correlated with disease severity(r=0.535,P<0.01),was negatively correlated with the level of urinary protein(r=-0745,P<0.001);After the expression of miR-320c was up-regulated in human umbilical vein endothelial cells transfected with the mimics,the expression of VEGF protein was significantly decreased(P<0.05).Conclusion:There are differences in exosome levels between patients with normal pregnancy and severe preeclampsia.Gestational increase and disease aggravation both promote exosome secretion in the body,and exosome components are changed in pathological pregnancy.In this study,the expression of miR-320c in exosomes of preeclampsia patients was decreased,and the expression of miR-320c in HUVEC was up-regulated,and the expression of VEGF protein was significantly decreased.Confirmed preeclampsia secrete body outside the change of the active component with preeclampsia patients with systemic vascular endothelial injury and inflammation reaction activation,body composition,release signal molecule component change of a large number of active vesicles,transported to the target cell injury and the receptor interaction in preeclampsia vascular injury and repair,hypoxia organizations play an important role in angiogenesis.更多还原