發(fā)布時(shí)間：2018-03-10 12:02

  本文選題：早發(fā)型重度子癇前期　切入點(diǎn)：合體滋養(yǎng)細(xì)胞微粒　出處：《第三軍醫(yī)大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景:妊娠期高血壓疾病是妊娠期特有的一組疾病,我國(guó)的發(fā)病率約5%-8%,是導(dǎo)致孕產(chǎn)婦死亡的重要原因,約占妊娠相關(guān)死亡總數(shù)的10%-16%[1]。子癇前期(pre-eclampsia,PE)/子癇(eclampsia)是妊娠期高血壓疾病中最嚴(yán)重的類(lèi)型,特指妊娠20周后新出現(xiàn)的高血壓和蛋白尿,常繼發(fā)母體多器官功能受損及胎兒并發(fā)癥。按照病情的輕重分為輕度子癇前期(mild pre-eclampsia,m PE)和重度子癇前期(severe pre-eclampsia,sPE),以妊娠34周發(fā)病為界限,又分為早發(fā)型(㩳34周)和晚發(fā)型(≥34周)兩種亞型,尤其以早發(fā)型重度子癇前期(early-onset sPE)危害性大,嚴(yán)重威脅母兒近期及遠(yuǎn)期的健康和生存質(zhì)量。然而,PE的確切病因和發(fā)病機(jī)制仍未闡明,目前缺乏簡(jiǎn)便、有效、可靠和經(jīng)濟(jì)的疾病預(yù)測(cè)方法,臨床治療陷于被動(dòng),長(zhǎng)期以來(lái)以“對(duì)癥治療結(jié)合終止妊娠”為主,母兒結(jié)局并不樂(lè)觀。新的治療策略及方案有賴于對(duì)PE病因和發(fā)病機(jī)制的深入挖掘。學(xué)術(shù)界廣泛接納PE發(fā)病的“兩階段”假說(shuō)[2],以及近年來(lái)在此基礎(chǔ)上發(fā)展形成的“三階段”病因?qū)W說(shuō)[3,4]認(rèn)為胎盤(pán)源性不良因子的釋放是PE發(fā)病關(guān)鍵的病理生理環(huán)節(jié),血管內(nèi)皮損傷是發(fā)病的終末通路和中心環(huán)節(jié)。合體滋養(yǎng)細(xì)胞外囊泡(syncytiotrophoblast extracellular vesicles,STB-EVs)是自胎盤(pán)脫落直接進(jìn)入母體血循環(huán)的帶膜囊泡狀結(jié)構(gòu),主要包括合體滋養(yǎng)細(xì)胞微粒(syncytiotrophoblast microvesicles,STBM)和合體滋養(yǎng)細(xì)胞外泌體(syncytiotrophoblast exosomes,STBE),占血漿總微粒的1.5%-3%[5]。作為重要的胎盤(pán)源性因子,STB-EVs已被發(fā)現(xiàn)了二十余年,其釋放行為是正常妊娠過(guò)程中的一種生理現(xiàn)象,可調(diào)控母體適度的炎性反應(yīng)和生理性高凝狀態(tài)等,利于正常妊娠的維持。然而,當(dāng)胎盤(pán)釋放的STB-EVs在數(shù)量和質(zhì)量上發(fā)生變化時(shí),STB-EV具有損傷血管內(nèi)皮、促炎癥反應(yīng)、促凝血以及誘導(dǎo)免疫耐受失衡等特性,在PE的發(fā)生發(fā)展中發(fā)揮至關(guān)重要的作用[6-9]。既往研究認(rèn)為STB-EVs在數(shù)量上的差異性可能是其致病的重要原因,PE患者外周血中STB-EVs的數(shù)量顯著增多,但迄今為止大部分的體內(nèi)實(shí)驗(yàn)或體外實(shí)驗(yàn)多為現(xiàn)象的觀察與描述,較少涉及STB-EVs具體致病機(jī)制的深入探討,這可能與stb-evs的成分構(gòu)成不清、參與重要病理過(guò)程的關(guān)鍵致病因子不明有關(guān)。早發(fā)型重度子癇前期是一種胎盤(pán)源性疾病,其病因及發(fā)病機(jī)制不同于晚發(fā)型,更鮮有研究針對(duì)早發(fā)型重度子癇前期患者和正常妊娠婦女胎盤(pán)stb-evs成分構(gòu)成的對(duì)比分析,限制了stb-evs對(duì)于pe尤其是早發(fā)型spe致病機(jī)制的進(jìn)一步探索。生物體內(nèi)最終執(zhí)行功能的是蛋白質(zhì),pe患者體內(nèi)產(chǎn)生的stb-evs可能在“質(zhì)量”即蛋白質(zhì)成分構(gòu)成上不同于正常妊娠狀態(tài)[10]。因此全面解析pe及正常妊娠時(shí)stb-evs的差異蛋白質(zhì)表達(dá)譜,在此基礎(chǔ)上挖掘關(guān)鍵的致病因子,為進(jìn)一步探索stb-evs導(dǎo)致pe發(fā)病的具體機(jī)制、疾病的早期診斷及臨床療效的評(píng)估等奠定重要的研究基礎(chǔ)。此外,對(duì)stb-evs相關(guān)致病因子進(jìn)行干預(yù)和阻斷,也將為pe的臨床治療提供新的思路與策略。差異蛋白質(zhì)組學(xué)著重于篩選和鑒定不同種類(lèi)或不同狀態(tài)下各樣本間蛋白質(zhì)組的區(qū)別和變化,在疾病的早期診斷、病情進(jìn)展的監(jiān)控和臨床治療效果的判斷等方面具有光明的應(yīng)用前景。同位素標(biāo)記相對(duì)和絕對(duì)定量(isobarictagsforrelativeandabsolutequantitation,itraq)是近年來(lái)運(yùn)用廣泛的一種新型蛋白質(zhì)組學(xué)定量研究技術(shù),具有高通量、高靈敏度、低檢測(cè)限、分離能力強(qiáng)、分析范圍廣等特點(diǎn)。本研究采用itraq定量蛋白組學(xué)相關(guān)技術(shù)分析早發(fā)型重度子癇前期患者與正常妊娠婦女胎盤(pán)合體滋養(yǎng)細(xì)胞外囊泡的差異表達(dá)蛋白,并進(jìn)一步研究差異表達(dá)蛋白唾液酸結(jié)合的免疫球蛋白樣凝集素6(sialicacid-bindingig-likelectin6,siglec-6)在血管內(nèi)皮損傷中的作用及相關(guān)機(jī)制。材料與方法:1、獲取早發(fā)型重度子癇前期患者及正常妊娠婦女胎盤(pán)組織,采用絨毛組織塊培養(yǎng)結(jié)合四步差速/超速離心法體外制備胎盤(pán)stb-evs,應(yīng)用掃描電鏡、免疫電鏡及westernblotting方法進(jìn)行形態(tài)學(xué)及免疫來(lái)源的鑒定。2、通過(guò)itraq定量蛋白組學(xué)技術(shù)結(jié)合生物信息學(xué)方法分析早發(fā)型重度子癇前期患者及正常妊娠婦女胎盤(pán)stb-evs中差異表達(dá)的蛋白質(zhì),并應(yīng)用westernblotting方法對(duì)差異表達(dá)的蛋白進(jìn)行驗(yàn)證。3、獲取健康未孕婦女、早發(fā)型重度子癇前期患者及正常妊娠婦女分娩前及分娩后的血漿。通過(guò)westernblotting方法分析早發(fā)型重度子癇前期患者及正常妊娠婦女血漿中siglec-6蛋白的表達(dá)情況,并進(jìn)一步用elisa法測(cè)定siglec-6在健康未孕婦女、早發(fā)型重度子癇前期患者及正常妊娠婦女分娩前及分娩后血漿中的含量。4、以人臍靜脈內(nèi)皮細(xì)胞huvec為實(shí)驗(yàn)平臺(tái),利用cck-8細(xì)胞增殖實(shí)驗(yàn)檢測(cè)siglec-6在不同的處理濃度及處理時(shí)間條件下對(duì)huvec細(xì)胞增殖的反應(yīng);利用細(xì)胞劃痕實(shí)驗(yàn)和transwell小室遷移實(shí)驗(yàn)檢測(cè)不同的siglec-6處理對(duì)huvec細(xì)胞水平遷移和固有遷移能力的影響;利用tunel法檢測(cè)siglec-6處理對(duì)huvec細(xì)胞凋亡的影響;利用elisa法測(cè)定不同的siglec-6處理對(duì)huvec細(xì)胞分泌il-6、tnf-α及vegf的影響;采用westernblotting檢測(cè)siglec-6處理后huvec細(xì)胞中磷脂酰肌醇3激酶(phosphoinositide3-kinase,pi3k)的表達(dá)及蛋白激酶b(proteinkinaseb,pkb/akt)磷酸化水平的變化情況。結(jié)果:1、掃描電鏡和免疫電鏡顯示stb-evs呈圓形、球形、狹長(zhǎng)型、杯形甚至不規(guī)則形,大小各異,多個(gè)囊泡聚集成團(tuán),囊泡大小在30nm-1700nm之間,與前期研究相吻合[9]。免疫電鏡證實(shí)了stb-evs的滋養(yǎng)細(xì)胞來(lái)源特性。westernblotting檢測(cè)表明stb-evs中存在hsp70、tsg101及flotillin-1等囊泡標(biāo)志蛋白。2、運(yùn)用itraq定量蛋白質(zhì)組學(xué)相關(guān)技術(shù)對(duì)早發(fā)型重度子癇前期患者與正常妊娠婦女胎盤(pán)stb-evs進(jìn)行差異蛋白質(zhì)分析,共鑒定出194個(gè)差異表達(dá)的蛋白,其中122個(gè)差異蛋白在病例組表達(dá)上調(diào),72個(gè)表達(dá)下調(diào)。進(jìn)一步的生物信息學(xué)分析表明這些差異表達(dá)蛋白參與細(xì)胞組成、生物過(guò)程和分子功能分別富集于線粒體、跨膜轉(zhuǎn)運(yùn)和跨膜轉(zhuǎn)運(yùn)蛋白活性方面。差異表達(dá)蛋白參與的主要代謝及信號(hào)通路為糖酵解/糖異生、檸檬酸循環(huán)、脂肪酸延伸、類(lèi)固醇激素生物合成以及氧化磷酸化。在早發(fā)型重度子癇前期組表達(dá)上調(diào)的差異表達(dá)蛋白涉及炎癥反應(yīng)、凝血過(guò)程、抗血管生成、免疫調(diào)節(jié)等子癇前期發(fā)病的主要病理生理過(guò)程,候選蛋白包括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是早發(fā)型重度子癇前期胎盤(pán)stb-evs中上調(diào)最為顯著的蛋白。westernblotting方法檢測(cè)差異表達(dá)蛋白cd63、s100-a8、siglec-6以及calnexin的表達(dá),驗(yàn)證了其與itraq定量分析的結(jié)果一致。3、westernblotting方法證實(shí)早發(fā)型重度子癇前期患者血漿中siglec-6蛋白的水平顯著高于正常妊娠婦女。elisa檢測(cè)siglec-6在健康未孕女性外周血中幾乎無(wú)表達(dá)(0.08±0.0007ng/ml),在未分娩的早發(fā)型重度子癇前期患者及正常妊娠婦女血漿中的含量分別為0.80±0.23ng/ml和0.21±0.04ng/ml,且分娩后siglec-6的表達(dá)迅速降低,分別為0.09±0.007ng/ml及0.09±0.01ng/ml,接近未孕水平。4、CCK-8細(xì)胞增殖實(shí)驗(yàn)表明Siglec-6處理可抑制人臍靜脈內(nèi)皮細(xì)胞HUVEC細(xì)胞的增殖,在測(cè)試的濃度和時(shí)間中,以2.0 ng/ml的Siglec-6處理48h對(duì)細(xì)胞增殖的抑制作用最強(qiáng)(P0.05);Transwell小室遷移實(shí)驗(yàn)顯示2.0 ng/ml的Siglec-6顯著抑制HUVEC細(xì)胞在水平方向遷移能力(P0.05);細(xì)胞劃痕實(shí)驗(yàn)顯示2.0 ng/ml的Siglec-6可抑制HUVEC細(xì)胞的固有遷移能力(P0.05),濃度升高至5.0 ng/ml時(shí)對(duì)HUVEC細(xì)胞固有遷移能力的抑制作用更明顯(P0.05);TUNEL法檢測(cè)結(jié)果表明2.0 ng/ml的Siglec-6可促進(jìn)HUVEC細(xì)胞的凋亡(P0.05);ELISA測(cè)定結(jié)果顯示:Siglec-6以劑量和時(shí)間依賴模式促進(jìn)HUVEC細(xì)胞分泌IL-6與TNF-α(P0.05),Siglec-6抑制HUVEC細(xì)胞分泌VEGF(P0.05),且這種抑制作用與Siglec-6的劑量和作用時(shí)間不相關(guān)。Western blotting檢測(cè)結(jié)果顯示,Siglec-6可致HUVEC細(xì)胞中PI3K的表達(dá)以及AKT磷酸化水平降低(P0.05)。結(jié)論:1、早發(fā)型重度子癇前期與正常妊娠婦女胎盤(pán)STB-EVs的蛋白質(zhì)表達(dá)譜存在差異。篩選發(fā)現(xiàn)Siglec-6是早發(fā)型重度子癇前期STB-EVs中上調(diào)最顯著的差異表達(dá)蛋白。2、Siglec-6在早發(fā)型重度子癇前期患者外周血中的表達(dá)水平顯著高于正常妊娠組,其含量約為正常妊娠婦女的4倍,且分娩后表達(dá)迅速降低,接近未孕水平。血漿中Siglec-6的水平變化為臨床終止妊娠是早發(fā)型重度子癇前期唯一有效的治療手段提供了實(shí)驗(yàn)佐證。3、Siglec-6可能通過(guò)調(diào)控PI3K-AKT信號(hào)通路,抑制血管內(nèi)皮細(xì)胞的增殖和遷移、促進(jìn)血管內(nèi)皮細(xì)胞的凋亡及炎性因子IL-6及TNF-α的分泌、抑制VEGF的分泌,導(dǎo)致血管內(nèi)皮損傷。Siglec-6可能是誘發(fā)早發(fā)型重度子癇前期發(fā)病的關(guān)鍵蛋白之一。
[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鏂規(guī)硶瀵瑰樊寮傝〃杈劇殑铔嬬櫧榪涜楠岃瘉.3,鑾峰彇鍋ュ悍鏈瓡濡囧コ,鏃╁彂鍨嬮噸搴﹀瓙鐥墠鏈熸?zhèn)ｈ�呭強(qiáng)姝ｅ父濡婂濡囧コ鍒嗗ī鍓嶅強(qiáng)鍒嗗ī鍚庣殑琛，

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