早发型重度子痫前期合体滋养细胞外囊泡的差异蛋白质组学研究

发布时间：2018-03-10 12:02

  本文选题：早发型重度子痫前期　切入点：合体滋养细胞微粒　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：背景:妊娠期高血压疾病是妊娠期特有的一组疾病,我国的发病率约5%-8%,是导致孕产妇死亡的重要原因,约占妊娠相关死亡总数的10%-16%[1]。子痫前期(pre-eclampsia,PE)/子痫(eclampsia)是妊娠期高血压疾病中最严重的类型,特指妊娠20周后新出现的高血压和蛋白尿,常继发母体多器官功能受损及胎儿并发症。按照病情的轻重分为轻度子痫前期(mild pre-eclampsia,m PE)和重度子痫前期(severe pre-eclampsia,sPE),以妊娠34周发病为界限,又分为早发型(�d34周)和晚发型(≥34周)两种亚型,尤其以早发型重度子痫前期(early-onset sPE)危害性大,严重威胁母儿近期及远期的健康和生存质量。然而,PE的确切病因和发病机制仍未阐明,目前缺乏简便、有效、可靠和经济的疾病预测方法,临床治疗陷于被动,长期以来以“对症治疗结合终止妊娠”为主,母儿结局并不乐观。新的治疗策略及方案有赖于对PE病因和发病机制的深入挖掘。学术界广泛接纳PE发病的“两阶段”假说[2],以及近年来在此基础上发展形成的“三阶段”病因学说[3,4]认为胎盘源性不良因子的释放是PE发病关键的病理生理环节,血管内皮损伤是发病的终末通路和中心环节。合体滋养细胞外囊泡(syncytiotrophoblast extracellular vesicles,STB-EVs)是自胎盘脱落直接进入母体血循环的带膜囊泡状结构,主要包括合体滋养细胞微粒(syncytiotrophoblast microvesicles,STBM)和合体滋养细胞外泌体(syncytiotrophoblast exosomes,STBE),占血浆总微粒的1.5%-3%[5]。作为重要的胎盘源性因子,STB-EVs已被发现了二十余年,其释放行为是正常妊娠过程中的一种生理现象,可调控母体适度的炎性反应和生理性高凝状态等,利于正常妊娠的维持。然而,当胎盘释放的STB-EVs在数量和质量上发生变化时,STB-EV具有损伤血管内皮、促炎症反应、促凝血以及诱导免疫耐受失衡等特性,在PE的发生发展中发挥至关重要的作用[6-9]。既往研究认为STB-EVs在数量上的差异性可能是其致病的重要原因,PE患者外周血中STB-EVs的数量显著增多,但迄今为止大部分的体内实验或体外实验多为现象的观察与描述,较少涉及STB-EVs具体致病机制的深入探讨,这可能与stb-evs的成分构成不清、参与重要病理过程的关键致病因子不明有关。早发型重度子痫前期是一种胎盘源性疾病,其病因及发病机制不同于晚发型,更鲜有研究针对早发型重度子痫前期患者和正常妊娠妇女胎盘stb-evs成分构成的对比分析,限制了stb-evs对于pe尤其是早发型spe致病机制的进一步探索。生物体内最终执行功能的是蛋白质,pe患者体内产生的stb-evs可能在“质量”即蛋白质成分构成上不同于正常妊娠状态[10]。因此全面解析pe及正常妊娠时stb-evs的差异蛋白质表达谱,在此基础上挖掘关键的致病因子,为进一步探索stb-evs导致pe发病的具体机制、疾病的早期诊断及临床疗效的评估等奠定重要的研究基础。此外,对stb-evs相关致病因子进行干预和阻断,也将为pe的临床治疗提供新的思路与策略。差异蛋白质组学着重于筛选和鉴定不同种类或不同状态下各样本间蛋白质组的区别和变化,在疾病的早期诊断、病情进展的监控和临床治疗效果的判断等方面具有光明的应用前景。同位素标记相对和绝对定量(isobarictagsforrelativeandabsolutequantitation,itraq)是近年来运用广泛的一种新型蛋白质组学定量研究技术,具有高通量、高灵敏度、低检测限、分离能力强、分析范围广等特点。本研究采用itraq定量蛋白组学相关技术分析早发型重度子痫前期患者与正常妊娠妇女胎盘合体滋养细胞外囊泡的差异表达蛋白,并进一步研究差异表达蛋白唾液酸结合的免疫球蛋白样凝集素6(sialicacid-bindingig-likelectin6,siglec-6)在血管内皮损伤中的作用及相关机制。材料与方法:1、获取早发型重度子痫前期患者及正常妊娠妇女胎盘组织,采用绒毛组织块培养结合四步差速/超速离心法体外制备胎盘stb-evs,应用扫描电镜、免疫电镜及westernblotting方法进行形态学及免疫来源的鉴定。2、通过itraq定量蛋白组学技术结合生物信息学方法分析早发型重度子痫前期患者及正常妊娠妇女胎盘stb-evs中差异表达的蛋白质,并应用westernblotting方法对差异表达的蛋白进行验证。3、获取健康未孕妇女、早发型重度子痫前期患者及正常妊娠妇女分娩前及分娩后的血浆。通过westernblotting方法分析早发型重度子痫前期患者及正常妊娠妇女血浆中siglec-6蛋白的表达情况,并进一步用elisa法测定siglec-6在健康未孕妇女、早发型重度子痫前期患者及正常妊娠妇女分娩前及分娩后血浆中的含量。4、以人脐静脉内皮细胞huvec为实验平台,利用cck-8细胞增殖实验检测siglec-6在不同的处理浓度及处理时间条件下对huvec细胞增殖的反应;利用细胞划痕实验和transwell小室迁移实验检测不同的siglec-6处理对huvec细胞水平迁移和固有迁移能力的影响;利用tunel法检测siglec-6处理对huvec细胞凋亡的影响;利用elisa法测定不同的siglec-6处理对huvec细胞分泌il-6、tnf-α及vegf的影响;采用westernblotting检测siglec-6处理后huvec细胞中磷脂酰肌醇3激酶(phosphoinositide3-kinase,pi3k)的表达及蛋白激酶b(proteinkinaseb,pkb/akt)磷酸化水平的变化情况。结果:1、扫描电镜和免疫电镜显示stb-evs呈圆形、球形、狭长型、杯形甚至不规则形,大小各异,多个囊泡聚集成团,囊泡大小在30nm-1700nm之间,与前期研究相吻合[9]。免疫电镜证实了stb-evs的滋养细胞来源特性。westernblotting检测表明stb-evs中存在hsp70、tsg101及flotillin-1等囊泡标志蛋白。2、运用itraq定量蛋白质组学相关技术对早发型重度子痫前期患者与正常妊娠妇女胎盘stb-evs进行差异蛋白质分析,共鉴定出194个差异表达的蛋白,其中122个差异蛋白在病例组表达上调,72个表达下调。进一步的生物信息学分析表明这些差异表达蛋白参与细胞组成、生物过程和分子功能分别富集于线粒体、跨膜转运和跨膜转运蛋白活性方面。差异表达蛋白参与的主要代谢及信号通路为糖酵解/糖异生、柠檬酸循环、脂肪酸延伸、类固醇激素生物合成以及氧化磷酸化。在早发型重度子痫前期组表达上调的差异表达蛋白涉及炎症反应、凝血过程、抗血管生成、免疫调节等子痫前期发病的主要病理生理过程,候选蛋白包括s100-a8,c4b-b,cd63,atpsynthasesubunitbeta,cdnaflj14908fis,endoglin,interleukin-27subunitbeta,f11receptor,isoformcra_a,dynamin-2,proteinsec13homolog,calnexin,serpinb9,stomatin-likeprotein2,phosphoinositide3-kinaseadapterprotein1和dolichyl-diphosphooligosaccharide-proteinglycosyltransferase48kdasubunit。siglec-6是早发型重度子痫前期胎盘stb-evs中上调最为显著的蛋白。westernblotting方法检测差异表达蛋白cd63、s100-a8、siglec-6以及calnexin的表达,验证了其与itraq定量分析的结果一致。3、westernblotting方法证实早发型重度子痫前期患者血浆中siglec-6蛋白的水平显著高于正常妊娠妇女。elisa检测siglec-6在健康未孕女性外周血中几乎无表达(0.08±0.0007ng/ml),在未分娩的早发型重度子痫前期患者及正常妊娠妇女血浆中的含量分别为0.80±0.23ng/ml和0.21±0.04ng/ml,且分娩后siglec-6的表达迅速降低,分别为0.09±0.007ng/ml及0.09±0.01ng/ml,接近未孕水平。4、CCK-8细胞增殖实验表明Siglec-6处理可抑制人脐静脉内皮细胞HUVEC细胞的增殖,在测试的浓度和时间中,以2.0 ng/ml的Siglec-6处理48h对细胞增殖的抑制作用最强(P0.05);Transwell小室迁移实验显示2.0 ng/ml的Siglec-6显著抑制HUVEC细胞在水平方向迁移能力(P0.05);细胞划痕实验显示2.0 ng/ml的Siglec-6可抑制HUVEC细胞的固有迁移能力(P0.05),浓度升高至5.0 ng/ml时对HUVEC细胞固有迁移能力的抑制作用更明显(P0.05);TUNEL法检测结果表明2.0 ng/ml的Siglec-6可促进HUVEC细胞的凋亡(P0.05);ELISA测定结果显示:Siglec-6以剂量和时间依赖模式促进HUVEC细胞分泌IL-6与TNF-α(P0.05),Siglec-6抑制HUVEC细胞分泌VEGF(P0.05),且这种抑制作用与Siglec-6的剂量和作用时间不相关。Western blotting检测结果显示,Siglec-6可致HUVEC细胞中PI3K的表达以及AKT磷酸化水平降低(P0.05)。结论:1、早发型重度子痫前期与正常妊娠妇女胎盘STB-EVs的蛋白质表达谱存在差异。筛选发现Siglec-6是早发型重度子痫前期STB-EVs中上调最显著的差异表达蛋白。2、Siglec-6在早发型重度子痫前期患者外周血中的表达水平显著高于正常妊娠组,其含量约为正常妊娠妇女的4倍,且分娩后表达迅速降低,接近未孕水平。血浆中Siglec-6的水平变化为临床终止妊娠是早发型重度子痫前期唯一有效的治疗手段提供了实验佐证。3、Siglec-6可能通过调控PI3K-AKT信号通路,抑制血管内皮细胞的增殖和迁移、促进血管内皮细胞的凋亡及炎性因子IL-6及TNF-α的分泌、抑制VEGF的分泌,导致血管内皮损伤。Siglec-6可能是诱发早发型重度子痫前期发病的关键蛋白之一。
[Abstract]:Background: hypertensive disorders in pregnancy is a pregnancy specific disorder, the incidence of about 5%-8%, is an important cause of maternal death, pregnancy related deaths accounted for about 10%-16%[1]. of the total number of preeclampsia / eclampsia (pre-eclampsia, PE) (eclampsia) is the most serious type of hypertensive disorders in pregnancy, especially during pregnancy 20 weeks of new hypertension and proteinuria, Chang Jifa maternal and fetal complications of multiple organ dysfunction. According to the severity of the disease is divided into mild preeclampsia (mild pre-eclampsia, m PE) and severe preeclampsia (severe pre-eclampsia, sPE), with 34 weeks of pregnancy onset limits is divided into early onset (34 weeks?) and late onset (34 weeks) two subtypes, particularly in the early onset of severe preeclampsia (early-onset sPE) harm, a serious threat to maternal and fetal short-term and long-term health and quality of life. However, the exact etiology and pathogenesis of PE The mechanism has not been elucidated, the current lack of simple, effective, reliable and economical method to predict the disease, clinical treatment in a passive, long time to symptomatic treatment with termination of pregnancy, perinatal outcomes and treatment strategies is not optimistic. The new scheme depends on deep mining PE etiology and pathogenesis. The academic circles widely accepted the incidence of PE in the "two stage" hypothesis of [2], and in recent years on the basis of the development of the formation of "three stages" of the etiology of [3,4] that placenta derived adversefactors release is key part of the pathophysiology of the pathogenesis of PE, vascular endothelial injury is the final pathway and central part of the pathogenesis. The syncytiotrophoblast of extracellular vesicles (syncytiotrophoblast extracellular vesicles, STB-EVs) is from placenta shedding directly into the maternal blood circulation with membrane vesicles, including syncytiotrophoblast microparticles (syncytiotrophoblast micro Vesicles, STBM) and syneytiotrophoblast exosome (syncytiotrophoblast exosomes, STBE), accounting for total plasma particles 1.5%-3%[5]. as placenta derived factor, STB-EVs has been found in more than twenty years, its release behavior is a physiological process of normal pregnancy phenomenon, can regulate maternal moderate inflammatory reaction and the physiological hypercoagulable state, conducive to the maintenance of normal pregnancy. However, when the placenta release STB-EVs changes in the quantity and quality of STB-EV, with the injury of vascular endothelium and proinflammatory and procoagulant and induce immune tolerance by unbalanced characteristics in the occurrence and development of PE play the role of [6-9]. in critical thought the difference in the number of STB-EVs may be an important cause of the disease, the number of STB-EVs in peripheral blood of patients with PE increased significantly, but so far most of the experiments in vivo or in vitro for Observation and description of the phenomenon, in-depth study of STB-EVs less involved in specific pathogenic mechanisms, which may be related to the composition of stb-evs is not clear, the key pathogenic factors involved in the pathological process of the unknown. An early onset of severe preeclampsia is a placenta derived disease, its etiology and pathogenesis is different from the late onset, more comparative analysis few studies on patients with early onset severe preeclampsia and normal pregnant women placental stb-evs components, especially stb-evs limits the further exploration of the pathogenesis of early-onset SPE for PE. The executive function is the final organism protein produced in patients with PE stb-evs in "quality" is the protein composition of different proteins in the state of the [10]. spectrum of normal pregnancy and normal pregnancy so comprehensive analysis of PE stb-evs, mining the key pathogenic factors on the basis of this, for a Stb-evs causes the concrete step to explore the pathogenesis of PE, lay the foundation of important research and clinical evaluation of early diagnosis of disease. In addition, stb-evs related to the pathogenic factors of intervention and blocking, will also provide new ideas and methods for clinical treatment of PE. Differential proteomics focuses on the screening and identification of different species or under the condition of different differences and changes in the proteome of each sample, in the early diagnosis of the disease and has a bright future in application of monitoring progress and clinical evaluation of the therapeutic effect. Isobaric tags for relative and absolute (isobarictagsforrelativeandabsolutequantitation, iTRAQ) is widely used in recent years is a new type of quantitative proteomics research technology that is a high throughput, high sensitivity, low detection limit, strong separation ability, analysis of the characteristics of a wide range. This study used iTRAQ quantitative proteomics The related technical analysis of early onset of severe preeclampsia patients and normal pregnant women were vesicle syncytiotrophoblast expression protein, and further study the differential expression of immunoglobulin like sialic acid binding lectin protein 6 (sialicacid-bindingig-likelectin6, siglec-6) in the vascular endothelial injury and related mechanisms. Materials and methods: 1, access early onset severe preeclampsia and normal pregnancy placenta tissue, the villus tissue culture with four step / differential centrifugation in vitro preparation of placenta stb-evs, using scanning electron microscopy, immunoelectron microscopic identification of.2 and westernblotting method of morphology and immune source, through the iTRAQ quantitative proteomics technique combined with bioinformatics analysis of early onset severe preeclampsia and normal pregnant women placental protein differences in stb-evs expression, and application of W esternblotting鏂规硶瀵瑰樊寮傝〃杈剧殑铔嬬櫧杩涜�岄獙璇�.3,鑾峰彇鍋ュ悍鏈�瀛曞�囧コ,鏃╁彂鍨嬮噸搴﹀瓙鐥�鍓嶆湡鎮ｈ�呭強姝ｅ父濡婂�犲�囧コ鍒嗗ī鍓嶅強鍒嗗ī鍚庣殑琛，

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